Process for the Purification of Recombinant Albumin

ABSTRACT

A process is provided for the preparation of a highly pure recombinant albumin solution having a nickel ion content of less than 100 ng per gram of albumin. The process comprises subjecting a recombinant albumin to a series of chromatography, concentration, and diafiltration steps.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/890,297,Jan. 4, 2002, which is a national phase application filed frominternational patent application no. PCT/GB00/00257, filed Jan. 31,2000, which claims the benefit of Great Britain patent application no.9902000.0, filed Jan. 30, 1999, which applications are herebyincorporated herein by reference in their entirety.

The present invention relates to a process for purifying the proteinhuman serum albumin (HSA) extracted from serum or plasma, or recombinanthuman albumin (rHA) produced by transforming or transfecting an organismwith a nucleotide coding sequence encoding the amino acid sequence ofhuman serum albumin, including rHA produced using transgenic animals orplants. In this specification, the term “albumin” refers generically toHSA and/or rHA.

BACKGROUND OF THE INVENTION

Albumin is used to treat patients with severe burns, shock or bloodloss. It is also used to supplement media used for growing highereukaryotic cells and as an excipient for pharmacologically activecompounds, many of which need to be stabilised. At present, the demandfor the product is satisfied by albumin extracted from human blood.Examples of extraction and separation techniques include those disclosedin: JP 03/258 728 on the use of a cation exchanger; EP 428 758 oil theuse of anion exchange; and EP 452 753 on the use of heating, adding saltand diafiltering.

The production of rHA in micro-organisms has been disclosed in EP 330451 so and EP 361 991. Purification techniques for rHA have beendisclosed in: WO 92/04367, removal of matrix-derived dye; EP 464 590,removal of yeast-derived colorants; EP 319 067, alkaline precipitationand subsequent application of the rHA to a lipophilic phase; and WO96/37515, which contains several complete purification processes.

The present invention represents the result of intensive development ofthe processes described in WO 96/37515 and that of U.S. Pat. No.5,728,553, incorporated herein by reference.

SUMMARY OF THE INVENTION

A first aspect of the present invention provides a process for purifyingan albumin solution, the process comprising the step of subjecting afirst albumin solution of pH8.0-9.5, and having a conductivity in therange of 1 to 75 mS·cm⁻¹, to an affinity chromatography step which isrun in negative mode with respect to the albumin and which utilises anaffinity matrix comprising immobilised dihydroxyboryl groups, therebyobtaining a purified albumin solution.

Preferably, the pH of the first albumin solution is pH8.0-9.0, and morepreferably pH8.3-pH8.6. It is preferred that the first albumin solutionis buffered with a buffer having a pH within the aforementioned pHranges.

Preferably, the buffer comprises an amino acid at a concentration of10-500 mM, preferably 25-200 mM, and more preferably 50-150 mM.Preferably the amino acid is glycine.

Preferably, the buffer comprises a monovalent cation at a concentrationof 0-500 mM, preferably 25-200 mM, and more preferably 50-150 mM.Preferably, the monovalent cation is sodium, preferably in the form ofNaCl. Accordingly, in a preferred embodiment the buffer comprises NaClat a concentration of 0-500 mM, preferably 25-200 mM, and morepreferably 50-150 mM.

Preferably, the buffer comprises a divalent cation at a concentration of5-250 mM, preferably 10-100 mM. Preferably, the divalent cation iscalcium, preferably in the form of CaCl₂. Accordingly, in a preferredembodiment the buffer comprises CaCl₂ at a concentration of 5-250 mM,preferably 10-100 mM.

In a particularly preferred embodiment the first albumin solution and/orbuffer comprises about 100 mM glycine, about 100 mM NaCl and about 50 mMCaCl₂.

Preferably, the conductivity of the first albumin solution and/or bufferis 10-50 mS·cm⁻¹ and more preferably 18-22 mS·cm⁻¹.

Advantageously, the concentration of the albumin in the first albuminsolution is in the range of 20-120 g·L⁻¹, preferably 70-120 g·L⁻¹, andmore preferably 100±10 g·L⁻¹. Preferably, the albumin is loaded in lessthan 0.5 column volumes, more preferably in less than 0.35 columnvolumes.

Suitably, the matrix comprises a boronic acid. The term “acid” as usedherein includes the salts thereof. Advantageously, the boronic acid isbonded via a triazine or a substituted triazine, for example to formmonoborotriazine or diborotriazine, to a support such as agarose.Preferably, the boronic acid is aminophenylboronic acid.

Publications that cover alternatives to phenylboronate, such asaliphatic and substituted aromatic ligands, include Adamek, V. et al(1992) J. Chrom. 625, 91-99, Singhal, R E P et al (1991) J. Chrom 543,17-38 and Liu, X. et al (1994) 687, 61-69.

Suitably, following the affinity chromatography step the purifiedalbumin solution is subjected to further purification, preferablyfurther chromatographic purification. Preferably, the albumin is furtherpurified using cation exchange chromatography and/or anion exchangechromatography. The order of the cation and anion exchange steps can beinter-changed while still performing their purification objectives. Froman operational point of view, a better integrated process is cationexchange chromatography followed by anion exchange chromatography.

Suitably, the purified albumin solution produced according to theprocess of the first aspect of the present invention undergoes one ormore of, buffer exchange; concentration; dilution; dialysis;diafiltration, pH-adjustment (preferably to a pH greater than pH2.0 orpH4.0, and preferably to a pH less than pH10.0); treatment with areducing agent (eg as described in EP 570 916); decolouration treatment(eg with charcoal); heating (including sterilisation); cooling orconditioning; formulation for parenteral administration to a human; orplacing into a final container.

By parenteral administration we include intravenous administration,subcutaneous administration and intramuscular administration. Thealbumin may function as an excipient for a pharmacologically activeprotein, which may be administered parenterally.

A “final container” is one which leaves the manufacturer and isdistributed to customers such as hospitals and pharmacies.

A second aspect of the invention provides a process for purifying analbumin solution, the process comprising cation exchange chromatographyand anion exchange chromatography, wherein the thus purified albuminsolution optionally undergoes one or more of buffer exchange;concentration; dilution; dialysis; diafiltration; pH4-adjustment(preferably to a pH greater than pH2.0 or pH4.0, and preferably to a pHless than pH10.0); addition of reducing agent; decolouration treatment(eg with charcoal); heating (including sterilisation); cooling; orconditioning, but not further purification, in particular not furtherchromatographic purification, prior to being put into a final container.

The cation exchange chromatography step may follow the anion exchangechromatography step, or vice versa. Preferably, the cation exchangechromatography step is followed by the anion exchange chromatographystep.

Preferably, between the anion and cation exchange steps, there is nofurther purification step, although the albumin may be subjected tobuffer exchange etc. as noted above.

By conditioning, we mean any non-purifying handling step which improvesthe environment or condition of the albumin for the next step of theprocess or for final use. Conditioning can include the addition of analbumin stabiliser such as octanoate and/or other fatty acid, such as aC₆ or C₁₀ fatty acid, or sodium acetyl tryptophanate or mandelate.Conditioning can also include the addition of salts etc., and mayinvolve adjusting the conductivity of the albumin solution.

The cation exchange step of the first and second aspects of the presentinvention may be run in negative or positive mode with respect to thealbumin. In a preferred embodiment the cation exchange step is run innegative mode with respect to the albumin. Advantageously, theconditions are so chosen that glycosylated albumin binds more stronglyto the cation exchange material than non-glycosylated albumin.

The cation exchange chromatography step of the first and second aspectsof the present invention may utilise a commercial cation exchange matrixsuch as SP-Sepharose FF, SP-Spherosil, CM-Sepharose FF, CM-Cellulose,SE-Cellulose or S-Spheradex. Preferably, the cation exchange steputilises a matrix which comprises immobilised sulfopropyl substituentsas cation exchangers.

Preferably, the albumin solution which undergoes cation exchangechromatography has a pH of 4.5-6.0, more preferably a pH of 5.0-5.6, andyet more preferably a pH of 5.2-5.4.

Preferably, the albumin solution which undergoes cation exchangechromatography has an albumin concentration of 10-250 g·L⁻¹, preferably20-70 g·L⁻¹, and more preferably 50±10 g·L⁻¹.

Preferably, the albumin. solution which undergoes cation exchangechromatography has an octanoate ion concentration of 2-15 mM, preferably5-10 mM, and more preferably 6-9 mM.

Conveniently, prior to the cation exchange step, the albumin solutionundergoes one or more of the following processes: (i) pH-adjustment(preferably to a pH greater than pH2.0 or pH4.0, and preferably to a pHless than pH10.0); (ii) concentration; (iii) diafiltration; or (iv)conditioning by addition of a stabiliser such as octanoate and/or otherfatty acid, such as a C6 or C10 fatty acid, or sodium acetyltryptophanate or mandelate. Alternatively, or additionally, the albuminsolution undergoes one or more of buffer exchange; dilution; dialysis;diafiltration; treatment with a reducing agent; decolouration treatment(eg with charcoal); heating; cooling; or conditioning.

Generally, any modification involves additions, not removals.Preferably, the pH of the albumin solution is adjusted by the additionof acetic acid. Preferably, the albumin solution is concentrated byultrafiltration.

The anion exchange chromatography step of the first and second aspectsof the present invention may utilise a commercial anion exchange matrixsuch as Q Sepharose-FF, QMA-Spherosil, DEAE-Spherodex, Q-Hyper D,DEAL-cellulose, QAE-cellulose, or TMAE, DMAE, or DEAE Fractogel.Preferably, the anion exchange step utilises a matrix which comprisesimmobilised dialkylaminoalkyl (for example diethylaminoethyl)substituents as anion exchangers.

In one preferred embodiment the anion exchange chromatography step ofthe no first and second aspects of the present invention is run innegative mode with respect to the albumin.

Preferably, the albumin solution which undergoes negative mode anionexchange chromatography has a pH of 4.0-5.2, more preferably a pH of4.2-4.9, and yet more preferably a pH of 4.5-4.7.

Preferably, the albumin solution which undergoes anion exchangechromatography has a conductivity of less than 4.0 mS·cm⁻¹, and morepreferably a conductivity of 1.0±0.5 mS·cm⁻¹ and yet more preferably1.05±0.1 mS·cm⁻¹

Conveniently, prior to the anion exchange step, the albumin solutionundergoes pH adjustment and/or dilution with water. Preferably, the pHof the albumin solution is adjusted with acetic acid.

In another preferred embodiment the anion exchange chromatography stepof the first and second aspects of the present invention is run inpositive mode with respect to the albumin.

Suitably the albumin solution which undergoes positive mode anionexchange chromatography has a pH of 6.0-8.0, preferably a pH of 6.5-7.5,and yet more preferably a pH of 6.8 to 7.2. Preferably, the albuminsolution has been pH-adjusted using orthophosphate ions.

In one preferred embodiment the albumin concentration is 10-100 g·L⁻¹,more preferably 25-80 g·L⁻¹, and most preferably 30-60 g·L⁻¹.Preferably, the conductivity of the albumin solution is 1.0-2.0 mS·cm⁻¹,preferably 1.2-1.6 mS·cm⁻¹.

Suitably, the albumin is eluted from the anion exchanger with a bufferIC comprising 20-90 mM, preferably 30-70 mM and more preferably 35-65 mMof a phosphoric acid salt, for example sodium phosphate. Preferably, thealbumin is eluted from the anion exchanger with a buffer of pH6.0-8.0,preferably pH6.5-7.5.

It is particularly preferred that the processes of the first and secondaspects of the present invention are preceded by one or more of thefollowing steps: fermentation; primary separation; centrateconditioning; cation exchange chromatography, preferably usingsulfopropyl substituents as cation exchangers; anion exchangechromatography, preferably using diethylaminoalkyl substituents as anionexchangers; or affinity chromatography, preferably using an affinitymatrix which comprises an immobilised albumin-specific dye, preferably aCibacron Blue type of dye.

In a preferred embodiment of the present invention a process forpurifying albumin is provided which comprises the following steps:

(a) subjecting an albumin solution to a cation exchange chromatographystep run in positive mode with respect to the albumin;(b) collecting an albumin-containing cation exchange eluate;(c) subjecting the cation exchange eluate to an anion exchangechromatography step run in positive mode with respect to the albumin;(d) collecting an albumin-containing anion exchange eluate;(e) subjecting the anion exchange eluate to an affinity chromatographystep run in positive mode with respect to the albumin;(f) collecting an albumin-containing affinity chromatography eluate;(g) subjecting the affinity chromatography eluate to an affinitychromatography step run in negative mode with respect to the albumin andin positive mode with respect to glycoconjugates (glycosylated albuminand/or glycoproteins);(h) collecting the albumin-containing affinity chromatography flowthrough;(i) subjecting the affinity chromatography flow through to a cationexchange chromatography step run in negative mode with respect to thealbumin;(j) collecting the albumin-containing cation exchange flow through;(k) subjecting the cation exchange flow through to an anion exchangechromatography step run in negative mode or positive mode;(l) collecting the albumin-containing anion exchange flow throughwherein the anion exchange step is ran in negative mode; or eluting fromthe anion exchange matrix an anion exchange eluate wherein the anionexchange step is run in positive mode;and wherein any of the respective purification steps are optionallypreceded or followed by one or more of: buffer exchange; concentration;dilution; dialysis; diafiltration; pH-adjustment (preferably to a pHgreater than pH2.0 or pH4.0, and preferably to a pH less than pH10.0);treatment with a reducing agent; decolouration treatment (eg withcharcoal); heating (including sterilisation); cooling; or conditioning.

Accordingly, the purification steps may or may not be separated by oneor more of: buffer exchange; concentration; dilution; dialysis;diafiltration; pH-adjustment; treatment with a reducing agent;decolouration treatment; heating; cooling; or conditioning.

When any step is run in the negative mode for albumin, washings may becollected as well as flow through.

In another preferred embodiment of the present invention a process forpurifying albumin is provided which comprises the following steps.

(a) subjecting an albumin solution to a cation exchange chromatographystep run in positive mode with respect to the albumin;(b) collecting an albumin-containing cation exchange eluate;(c) subjecting the cation exchange eluate to an anion exchangechromatography step run in positive mode with respect to the albumin;(d) collecting an albumin-containing anion exchange eluate;(e) subjecting the anion exchange eluate to an affinity chromatographystep run in positive mode with respect to the albumin;(f) collecting an albumin-containing affinity chromatography eluate;(g) subjecting the affinity chromatography eluate to an affinitychromatography step run in negative mode with respect to the albumin andin positive mode with respect to glycoconjugates;(h) collecting the albumin-containing affinity chromatography flowthrough;(i) subjecting the affinity matrix flow through to an anion exchangechromatography step run in negative or positive mode with respect to thealbumin;(j) collecting the albumin-containing anion exchange flow throughwherein the anion exchange step is run in negative mode; or eluting fromthe anion exchange matrix an anion exchange eluate wherein the anionexchange step is run in positive mode;(k) subjecting the albumin solution purified by the anion exchangechromatography step to a cation exchange chromatography step run innegative mode with respect to the albumin;(l) collecting the albumin-containing cation exchange flow through;and wherein any of the respective purification steps are optionallypreceded or followed by one or more of buffer exchange; concentration;dilution; dialysis; diafiltration; pH-adjustment (preferably to a pHgreater than pH2.0 or pH4.0, and preferably to a pH less than pH10.0);treatment with a reducing agent; decolouration treatment (eg withcharcoal); heating (including sterilisation); cooling; or conditioning.

Accordingly, the purification steps may or may not be separated by oneor more of: buffer exchange; concentration; dilution; dialysis;diafiltration; pH-adjustment; treatment with a reducing agent;decolouration treatment; heating; cooling; or conditioning.

Preferably, prior to the positive mode cation exchange step of theinvention, the albumin solution is conditioned as above. Preferably, theoctanoate is added thereto to a final concentration of from about 1-10in M and the pH is adjusted to about 4.0-5.0.

Advantageously, the albumin retained in the positive cation exchangestep is washed with a high salt solution (eg 0.5-3.0M NaCl buffered atpH3.5 to 4.5, preferably at about pH 4.0, with 10-100 mM, preferably20-40 mM, for example 25-30 mM sodium acetate) before being eluted.

Preferably, the albumin is eluted in the cation exchange step using abuffer containing a compound having a specific affinity for albumin,especially an acid, for example octanoate or another fatty acid, forexample C₆ or C₁₀.

Suitably, the albumin is eluted from the anion exchanger, of the firstanion exchange step, with a buffer containing a high level (e.g. atleast 50 mM, preferably 50-200 mM, for example 80-150 mM) of a boricacid salt, for example sodium or potassium tetraborate.

Preferably, the positive mode affinity chromatography step uses a resincomprising an immobilised albumin-specific dye, such as a Cibacron Bluetype of dye, preferably immobilised on the resin via a spacer such as1,4-diaminobutane or another spacer of C₁₋₈, preferably C₁₋₆, eg C₁₋₅and Lost preferably C₄ length, preferably having α,ω-diaminosubstitution. Preferably, the matrix is the “Delta Blue Matrix” (DBA),prepared as described in WO 96/37515.

A third aspect of the present invention provides a process for reducingthe level of nickel ions in an albumin solution, the process comprisingsubjecting the albumin solution to a pH of 2.5 to 7.5, preferably2.5-6.0, and removing nickel ions. Preferably, the albumin solution issubjected to a pH of 4.0 to 7.5, preferably 4.0 to 6.0, more preferablypH4.0 to 5.5, yet more preferably pH4.0 to pH5.0, and most preferably topH4.0 to 4.5.

Preferably, the process of the third aspect of the invention comprisesdiafiltration against a buffer of pH2.5-6.0, or against a buffer havinga pH within one of the aforementioned pH ranges. Alternatively, nickelremoval can be achieved using gel permeation chromatography with abuffer having a pH within one of the above-listed pH ranges. Gelpermeation chromatography may be performed using Sephaeryl S200 HR.Preferably, the buffer comprises acetate and/or malate ions.Alternatively, there is sufficient buffering capacity from albumin toadjust the pH and perform diafiltration/gel permeation chromatographywith water.

The nickel ions can alternatively be chelated and removed from thealbumin. This can be achieved using a chelating agent such asiminodiacetic acid immobilised on Sepharose (Chelating Sepharose,Pharmacia) or another polymer (such as Chelex, Bio Rad Laboratories) ata low pH, preferably pH 4.0 to 6.0, more preferably pH4.0 to 4.5.

Preferably, when the product from the process of the third aspect of theinvention is subjected immediately to negative cation exchangechromatography it is preferred that the third aspect of the inventioncomprises subjecting the albumin solution to a pH of 5.0-5.6.Conversely, when the product from the process of the third aspect of theinvention is not subjected immediately to negative anion exchangechromatography it is preferred that the third aspect of the inventioncomprises subjecting the albumin solution to a pH of 4.3-4.9.

In a preferred embodiment of the first, second and third aspects of thepresent invention the initial albumin solution is derived from a fungalculture medium obtained by culturing a fungus transformed with analbumin-encoding nucleotide sequence in a fermentation medium, wherebysaid fungus expresses albumin and secretes it into the medium. Thefungus may be a filamentous fungus such as an Aspergillus species.Preferably, the fungus is a yeast. More preferably the fungus is of thegenus Saccharomyces (eg Saccharomyces cerevisiae), the genusKluyveromyces (eg Kluyveromyces lactis) or the genus Pichia (eg Pichiapastoris).

Preferably, at least some of the albumin purified in accordance with thefirst, second or third aspects of the present invention is produced by acell according to the fifth aspect of the invention or a processaccording to the sixth aspect of the invention.

A fourth aspect of the present invention provides an albumin solutionobtainable by a process according to any one of the preceding aspects ofthe present invention. Preferably, the albumin solution comprisesrecombinant albumin which exhibits one or more of the followingproperties:

(1) less than 0.5% (w/w) binds to Concanavalin A, preferably less than0.2% or 0.15%;(2) a glycation level of less than 0.6 moles hexose/mole of protein, andpreferably less than 0.10, 0.075 or 0.05 moles hexose/mole of protein.

A purified albumin solution prepared by a process of the presentinvention may be further processed according to its intended utility.For example, it may be ultrafiltered through an ultrafiltration membraneto obtain an ultrafiltration retentate having an albumin concentrationof at least about 10 g, preferably at least 40 g or more preferablyabout 80 g, albumin per litre, with the ultrafiltration retentate beingdiafiltered against at least 5 retentate equivalents of water.

A fifth aspect of the present invention provides a DNA sequence, plasmidor cell which comprises a recombinant albumin coding sequence whereinthe 3′ end of the recombinant albumin coding sequence comprises two ormore in-frame translation stop codons, and preferably three in-frametranslation stop codons.

The recombinant cells of the fifth aspect of the present invention maybe eukaryotic or prokaryotic. The recombinant cells may be bacteria (forexample E. coli or Bacillus subtilis), yeasts (for example a yeast ofthe genus Saccharomyces (eg S. cerevisiae), the genus Kluyveromyces (egK. lactis) or the genus Pichia (eg P. pastoris)), filamentous fungi (forexample Aspergillus), plants or plant cells, animals or animal cells(which may be transgenic) or insect cells.

A sixth aspect of the present invention provides a process for producingrecombinant albumin, the process comprising culturing a fungal cellexpressing a recombinant albumin coding sequence and obtaining thealbumin, wherein the cell has a genetic modification which causes thecell to have at least a reduced capacity of mannosylation of therecombinantly-expressed albumin and wherein the culture medium is atleast 1,000 L and is of pH6.0-6.8.

In the meaning of the present invention, genetic modification preferablymeans any suppression, substitution, deletion or addition of one or morebases or of a fragment of the fungal cell DNA sequences. Such geneticmodifications may be obtained in vitro (directly on isolated DNA) or insitu, for example by genetic engineering techniques or by exposing thefungal cells to mutagenic agents. Mutagenic agents include for examplephysical agents such as energetic rays X-rays, γ-rays, UV, etc.) orchemical agents capable of reacting with different functional groups ofDNA, such as alkylating agents (EMS, NQO, etc.) bisalkylating agents,intercalating agents, etc. Genetic modifications may also be obtained bygenetic disruption, for example according to the method disclosed byRothstein et al. [Meth. Enzymol. 194 (1991), 281-301]. According to thismethod, part or all of a gene is replaced, through homologousrecombination, by an in vitro modified version. Genetic modificationscan also be obtained by any mutational insertion on DNA sequences, suchas transposons, phages, etc.

It is known that certain modifications such as point mutations can bereversed or attenuated by cellular mechanisms. Such modifications maynot provide the most useful forms of modified fungal cells of thisinvention since their phenotypical properties may not be very stable.Accordingly, it is preferred that the genetic modification(s) are stablyinherited and/or are non-reverting and/or are non-leaky. Suchmodification(s) are generally obtained by a deletion or a genedisruption.

By a “leaky mutant” and grammatical variants thereof, we include mutantsthat result from a partial rather than a complete inactivation of thewild-type function.

The genetic modification(s) carried by the fungal cells of the inventionmay be located in a coding region of the DNA sequences of the celland/or in a region affecting the expression of a gene. Moreparticularly, said modification(s) will generally affect the codingregion or the region responsible for or involved in the expression ofone or more genes whose expression products are enzymes involved inmannosylation.

The reduced capacity of the fungal cells of the invention to mannosylateproteins may therefore result from the production of inactive enzymesdue to structural and/or conformational changes, from the production ofenzymes having altered biological properties, from the absence ofproduction of said enzymes, or from the production of said enzymes atlow levels.

The fungal cell mannosylation pathway involves attachment of a firstmannosyl residue to the hydroxyl group of seryl and/or threonyl aminoacids of proteins or peptides, and then the extension to O-linked di-and oligosaccharides by subsequent addition of mannosyl residues. Thefirst mannosyl residue is transferred from dolichol monophosphatemannose (Dol-P-Man) to the protein in the endoplasmic reticulum, and theadditional mannosyl residues are transferred from GPD-Man in the golgi.

In a preferred embodiment of the invention, the modified fungal cellscarry genetic modification(s) in at least one gene whose expressionproduct is involved in the attachment of a mannosyl residue to thehydroxyl group of seryl or threonyl amino acids.

In a another preferred embodiment of the invention, the modified fungalcells carry genetic modifications in at least one gene whose expressionproduct is involved in the transfer of a mannosyl residue from theDol-P-Man precursor to the hydroxyl group of seryl or threonyl aminoacids. Still more preferably, one of these genes is a PMT gene (eg PMT1,PMT2, PMT3, PMT4, PMT5, PMT6 or PMT7). Preferably the PMT gene is PMT1,PMT5 or PMT7.

WO 94/04687, incorporated herein by reference, describes the preparationof S. cerevisiae deficient in O-mannosylation activity. A S. cerevisiaecell deficient in O-mannosylation activity was prepared by genedisruption, by insertion of the URA3 gene into the HindIII restrictionsite of the PMT1 ORF. The resulting mutants were grown on YEPD (aboutpH6.95) or on minimal media+Ade, +Leu (about pH4.75, declining withyeast growth). Unexpectedly, we have found that the pHs of the growthmedia used in WO 94/04687 are not optimal for the large scale culture ofPMT mutants to produce secreted albumin. We have found that a growthmedium of pH6.0-6.8 is beneficial in terms of host cell integrity duringlarge scale fermentation.

In addition to modifications in a gene involved in the attachment ofmannosyl residues to the hydroxyl group of seryl or threonyl aminoacids, fungal cells of the invention may also carry modifications in thegenes involved in subsequent additions of mannosyl residues leading todi- or oligosaccharides, or in the synthesis of the mannosyl residuesdonor (Dol-P-Man).

Preferably, the fungal cell has a genetic modification within a PMT geneor a gene which affects the expression or product of a PMT gene. A genewhich affects the expression of a PMT gene may, for example, affect mRNAtranscript levels or PMT product levels.

The fungal cell of the sixth aspect of the present invention can bechosen from filamentous fungi and yeasts. Preferably, the cells areyeasts, for example a yeast of the genus Saccharomyces (eg S.cerevisiae), the genus Kluyveromyces (eg K. lactis) or the genus Pichia(eg P. pastoris).

Preferably, the fungal cell expressing the recombinant albumin codingsequence is cultured in a culture medium of at least 5,000 L, morepreferably at least 7,500 L.

Preferably, the fungal cell expressing the recombinant albumin codingsequence is cultured in a culture medium which is maintained in therange of pH6.2-6.7, more preferably pH6.3-6.5. Preferably, the pH of theculture medium is maintained using a pH controller set at a pH betweenpH6.3 and pH6.5, preferably at a pH: between 6.35 and 6.45 and morepreferably at about pH6.4. Preferably, the pH controller is controlledwithin 0.20 or 0.10 pH units of any pH value within any one of theaformentioned pH ranges or within 0.20 or 0.10 pH units of pH6.4.

In an alternative embodiment, the fungal cell is cultured in a culturemedium which is maintained in the range of pH5.30-pH5.90, preferablypH5.50-pH5.90, pH5.40-pH5.90 or pH5.40-5.60. Preferably, the lowercontrol set point is between pH5.40 and pH5.60, preferably betweenpH5.45 and pH5.55, and preferably the lower control set point is aboutpH5.50.

The present invention provides processes for the preparation of highlypurified albumin. The albumin is characterised by extremely low levelsof colorants. The term “colorant” as used herein means any compoundwhich colours albumin. For example, a pigment is a colorant which arisesfrom the organism, such as yeast, which is used to prepare recombinantalbumin, whereas a dye is a colorant which arises from chromatographicsteps to purify the albumin.

The albumin is also characterised by extremely low levels of, or bybeing essentially free of; aluminium, lactate, citrate, metals,non-albumin human proteins, such as immunoglobulins, pre-kallikreinactivator, transferrin,

-acid glycoprotein, haemoglobin and blood clotting factors, prokaryoticproteins, fragments of albumin, albumin aggregates or polymers, orendotoxin, billirubin, haem, yeast proteins, animal proteins andviruses. By essentially free is meant below detectable levels.

The albumin of the invention may be at least 99.5% monomeric anddimeric, preferably essentially 100% monomeric and dimeric. Up to 0.5%,preferably 0.2% trimer is tolerable but larger forms of albumin aregenerally absent. It may be further characterised by one or more of thefollowing characteristics. It has a nickel ion level of less than 100ng, based on one gram of albumin; a glycation level of less than 0.6,preferably less than 0.10, 0.075 or 0.05 moles hexose/mole protein asmeasured in the Amadori product assay; an intact, i.e. homogeneous,C-terminus; a content of conA-binding albumin of less than 0.5% (w/w),preferably less than 0.2% or 0.15%; a free thiol content of at least0.85 mole SH/mole protein; and substantially no C18 or C20 fatty acids.At least 99%, preferably at least 99.9%, by weight of the protein in thealbumin preparations purified by the process of the invention isalbumin. Such highly pure albumin is less likely to cause adverse sideeffects.

rHA purified according to the invention will generally be totally freeof serum-derived contaminants, since none are present in the startingmaterial.

In accordance with the present invention, highly pure albumin isobtained from an impure albumin solution. The process comprises one ormore of the following steps: culturing in a fermentation medium amicro-organism transformed with a nucleotide sequence encoding the aminoacid sequence of human albumin; preferably separating the micro-organismfrom the fermentation medium; conditioning the medium, if necessary, forfurther purification; passing the conditioned medium through threesuccessive chromatography steps; ultrafiltering/diafiltering theproduct; passing the ultrafiltered product through a furtherchromatography step; ultrafiltering/diafiltering again beforepurification through two further chromatographic steps; and finalultrafiltration/diafiltration.

Alternatively, instead of the fermentation medium, the impure albuminsolution may be a solution obtained from serum by any of the plethora ofextraction and purification techniques developed over the last 50 years,for example those disclosed in Stoltz et al (1991) Pharmaceut. Tech,Int. June 1991, 60-65 and More & Harvey (1991) in “Blood Separation andPlasma Fractionation” Ed. Harris, Wiley-Liss, 261-306.

In a further alternative, the albumin may be obtained from a transgenicanimal, such as goat, sheep or cattle, from, for instance, the milk orthe blood of the animal or, in the case of transgenic chicken, from theegg white.

In a still further alternative, the albumin may be obtained from atransgenic plant, such as tobacco, potato or corn (maize).

In instances where the albumin is purified from non-plasma sources,prior art purification processes lead to a relatively high level ofnickel ions. Albumin is known to have high affinity binding sites forcopper, nickel and zinc ions at the N-terminus of the molecule.Consequently, the albumin molecule effectively concentrates nickel ionsfrom the media used for cultivation and/or purification. Albuminpurified according to this invention has a surprisingly low level ofnickel ions.

Preceding or following any of the procedures of the present inventionthe albumin solution may undergo buffer exchange, concentration,dilution, heating (including sterilisation), cooling or may have saltsetc. added to the albumin solution which may, for example, condition oradjust the pH of the solution. Optionally, the albumin may be treatedwith a reducing agent or may undergo a decolouration step.

The final product may be formulated to give it added stability and maybe formulated according to its intended utility, eg it may be formulatedfor parenteral administration, preferably parenteral administration to ahuman. Suitably, the albumin undergoes sterilisation.

Preferably, the highly pure albumin product of the invention contains atleast 100 g, more preferably 1 kg or 10 kg of albumin, which may besplit between a plurality of vials.

The albumin of the present invention may be fulfil various roles inaddition to therapeutic use in the treatment of burns, shock or bloodloss. By way of example, it may be used as a final product excipient(e.g. in liquid formulations, freeze-dried formulations or formulationsfor inhalation), for stabilisation of other proteins duringpurification, in cell culture, viral production, gene therapy, in vitrofertilisation media, and for coating medical devices such as cannulae,catheters and vascular prostheses.

It should be appreciated that each aspect of the invention may becombined with one or more other aspects of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further illustrated in the appended drawings in which:

FIGS. 1 to 7 respectively show the construction of plasmids pAYE309,pAYE440, pAYE43S, pDB2241, pDB2242, pDB2243 and pDB2244; and

FIG. 8 shows electrospray mass spectrometry of conA-binding rHA fractionfrom rHA prepared according to the invention.

FIG. 9 shows the effect of pH and time on nickel removal from rHA byChelex™.

FIG. 10 (SEQ ID NO. 1) and 11 (SEQ ID NO. 2) represent two DNA sequenceswith homology to the protein encoding region Saccharomyces cerevisiaePMT1.

FIG. 12 (SEQ ID NO. 3), FIG. 13 (SEQ ID NO. 4), FIG. 14 (SEQ ID NO. 5),and FIG. 15 (SEQ ID NO. 6) represent four DNA sequences with homology tothe protein encoding region Saccharomyces cerevisiae PMT7.

FIG. 16 (SEQ ID NO. 7) and FIG. 17 (SEQ ID NO. 8) represent two DNAsequences with homology to the protein encoding region Saccharomycescerevisiae PMT5.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Whereas the processes of the present invention can be utilised to obtainhighly purified albumin from an impure albumin solution from a number ofsources, such as serum, it is particularly applicable to purifyingrecombinant human albumin (rHA). The albumin produced in accordance withthe invention may be any mammalian albumin, such as rat, bovine or ovinealbumin, but is preferably human albumin.

DNA encoding albumin may be expressed in a suitable host to producealbumin. Thus, DNA may be used in accordance with known techniques,appropriately modified in view of the teachings contained herein, toconstruct an expression vector, which is then used to transform anappropriate host cell for the expression and production of albumin.

The DNA encoding the albumin may be joined to a wide variety of otherDNA sequences for introduction into an appropriate host. The companionDNA will depend upon the nature of the host, the manner of theintroduction of the DNA into the host, and whether episomal maintenanceor integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognised bythe desired host, although such controls are generally available in theexpression vector. It is beneficial to incorporate more than one DNAsequence encoding a translational stop codon, such as UAA, UAG or UGA,in order to minimise translational read-through and thus avoid theproduction of elongated, non-natural fusion proteins. A DNA sequenceencoding the translation stop codon UAA is preferred. The vector is thenintroduced into the host through standard techniques, followed byselection for transformed host cells. Host cells so transformed are thencultured for a sufficient time and under appropriate conditions known tothose skilled in the art, and in view of the teachings disclosed herein,to permit the expression of the albumin, which can then be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae, Pichia pastoris and Kluyveromyces lactis), filamentous fungi(for example Aspergillus), plant cells, animal cells and insect cells.The preferred micro-organisms are the yeasts Saccharomyces cerevisiae,Kluyveromyces lactis and Pichia pastoris. It is particularlyadvantageous to use a yeast deficient in one or more protein mannosyltransferases involved in O-glycosylation of proteins, for instance bydisruption of the gene coding sequence.

The albumin protein sequence does not contain any sites for N-linkedglycosylation and has not been reported to be modified, in nature, byO-linked glycosylation. However, it has been found that rHA produced ina number of yeast species can be modified by O-linked glycosylation,generally involving mannose. The mannosylated albumin is able to bind tothe lectin Concanavalin A. The amount of mannosylated albumin producedby the yeast can be reduced by using a yeast strain deficient in one ormore of the PMT genes (WO 94/04687).

The most convenient way of achieving this is to create a yeast which hasa defect in its genome such that a reduced level of one of the Pmtproteins is produced. For example, there may be a deletion, insertion ortransposition in the coding sequence or the regulatory regions (or inanother gene regulating the expression of one of the PMT genes) suchthat little or no Pmt protein is produced. Alternatively, the yeastcould be transformed to produce an anti-Pmt agent, such as an anti-Pmtantibody.

To modify one of the PMT genes so that a reduced level of Pmt protein isproduced, site-directed mutagenesis or other known techniques can beemployed to create single or multiple mutations, such as replacements,insertions, deletions, and transpositions, as described in Botstein andShortle. “Strategies and Applications of In Vitro Mutagenesis”, Science,229: 193-210 (1985), which is incorporated herein by reference. Suitablemutations include chain termination mutations (clearly stop codonsintroduced near the 3′ end might have insufficient effect on the geneproduct to be of benefit; the person skilled in the art will readily beable to create a mutation in, say, the 5′ three quarters of the codingsequence), point mutations that alter the reading frame, small to largedeletions of coding sequence, mutations in the promoter or terminatorthat affect gene expression and mutations that de-stabilise the mRNA.Specific mutations can be introduced by an extension of the genedisruption technique known as gene transplacement (Winston, F. et al(1983) Methods Enzymol. 101, 211-228).

Generally, one uses a selectable marker to disrupt a gene sequence, butthis need not be the case, particularly if one can detect the disruptionevent phenotypically. In many instances the insertion of the interveningsequence will is be such that a stop codon is present in frame with thePmt sequence and the inserted coding sequence is not translated.Alternatively, the inserted sequence may be in a different reading frameto Pmt.

The gene may have one or more portions (optionally including regulatoryregions, up to the whole gene) excised or inverted, or it may have aportion inserted, in order to result in reduced production of proteinfrom one of the PMT loci and/or in the production of protein from one ofthe PMT loci having a reduced level of activity.

The PMT genes of Saccharomyces cerevisiae encode a family of seven(PMT1-PMT7) protein O-mannosyltransferases which vary in theirspecificity. These proteins are also known as dolicholphosphate-D-mannose: protein transferases, dolichyl-phosphate-D-mannose:protein O-D-mannosyltransferases or phosphomannose transferases(Gentzsch and Tanner, EMBO 15, 5752-5757, 1996, and references includedtherein). This family of integral membrane enzymes catalyses thetransfer of mannose, in the form of dolichyl phosphate mannose, onto thehydroxyl group of serine or threonine within the polypeptide chain,described by the following reaction:

Mg²⁺

Dol-P-Man+Protein (Ser/Thr)

Protein (Ser/Thr)−Man+Dol-P

The available evidence suggests that the synthesis of dolichyl phosphatemannose and the subsequent transfer of mannose to the protein occurs inthe endoplasmic reticulum.

It is clear that the enzymes of this family have different substrate(protein) no specificities (Gentzsch and Tanner (1997) Glycobiology 7,481-486). Five of seven test proteins were substrates for Pmt1p andPmt2p, the products of the PMT1 and PMT2 genes respectively, as shown bytheir under-glycosylation in pmt1 or pmt2 mutant Saccharomycescerevisiae strains. Another two test proteins were apparently unaffectedby either PMT1 or PMT2 mutations, but were under-glycosylated in pmt4mutant strain.

The 92 kD Pmt1p protein O-mannosyltransferase enzyme has been purifiedto homogeneity from solubilised Saccharomyces cerevisiae membranes(Strahl-Bolsinger and Tarner (1991) Eur. J. Biochem. 196, 185-190). Thegene encoding, for the Pmt1p (PMT1) has been cloned and sequenced. Thegene is located on chromosome IV and encodes a single polypeptide with aprimary sequence of 817 amino acids (Strahl-Bolsinger et al (1993)P.N.A.S. USA 90, 8164-8168). The sequence information of PMT1 (and otherPMT genes) may be used for the identification of relatedmannosyltransferases encoding genes in Saccharomyces cerevisiae.

The sequences shown in FIGS. 10 and 11 are homologous with the proteinencoding region Saccharomyces cerevisiae PMT1, the sequences shown inFIGS. 12 to 15 are homologous with the protein encoding regionSaccharomyces cerevisiae PMT7 and the sequences shown in FIGS. 16 to 17are homologous with the protein encoding region Saccharomyces cerevisiaePMT5 Persons skilled in the art will appreciate that any one of thesesequences may be used to identify (or disrupt) a Saccharomycescerevisiae mannosyltransferase gene. It will be appreciated thatfragments of the sequences represented in FIGS. 10 to 17 may similarlybe used, as may sequences which are homologous with the sequencesrepresented in FIGS. 10 to 17 and the fragments thereof. Techniques forgenerating homologous sequences are well known in the art.

It should be appreciated that by a homologous sequence, we includesequences having at least 70%, 80%, 90%, 95%, or 98% homology with asequence shown in any one of FIGS. 10 to 17, or with a fragment of asequence shown in any one of FIGS. 10 to 17.

Percent homology can be determined by, for example, by comparingsequence information using the GAP computer program, version 6.0described by Devereux et al. (Nucl. Acids res. 12:387, 1984) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilises the alignment method of Neddleman andWunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman(Adv. Appl. Math 2.482. 1981). The preferred default parameters for theGAP program include: (1) a unary comparison matrix (containing a valueof 1 for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Bribskov and Burgess, Nucl. Acids Res.14:6745, 1986 as described by Schwarts and Dayhoff, eds, Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

If a yeast other than S. cerevisiae is used, disruption of one or moreof the genes equivalent to the PMT genes of S. cerevisiae is alsobeneficial, eg in Pichia pastoris or Kluyveromyces lactis. The sequenceof PMT1 (or any other PMT gene) isolated from S. cerevisiae may be usedfor the identification or disruption of genes encoding similar enzymaticactivities in other fungal species. The cloning of the PMT1 homolog ofKluyveromyces lactis is described in WO 94/04687.

If a yeast other than S. cerevisiae is used, the sequences representedin FIGS. 10 to 17 may also be used to identify (or disrupt) a geneequivalent to a S. cerevisiae PMT gene. Persons skilled in the art willappreciate that fragments of the sequences represented in FIGS. 10 to 17may similarly be used, as may sequences which are homologous with thesequences represented in FIGS. 10 to 17 and the fragments thereof.

Methods for carrying out gene disruptions are described in theliterature, an example of which is described by Boehm et al. (Boehm, T.,Pirie-Shepherd, S., Trinh, L., Shiloach, J. and Folkman, J. 1999) Yeast15 563-572) which describes the use of the Saccharomyces cerevisiae SUC2gene as a marker Ranked by Pichia pastoris DNA specific to the targetgene. In the example of Pichia pastoris disruption, the SUC2 DNAsequence could be inserted at a position within any of the DNA sequencesrepresented in FIGS. 10 to 17. The yeast will advantageously have adeletion of the HSP150 and/or YAP3 genes as taught respectively in WO95/33833 and WO 95/23857.

In a preferred embodiment the yeast is transformed with an expressionplasmid based on the Saccharomyces cerevisiae 2 μm plasmid. At the timeof transforming the yeast, the plasmid contains bacterial replicationand selection sequences, which are excised, following transformation, byan internal recombination event in accordance with the teaching of EP286 424. The plasmid also contains an expression cassette comprising: ayeast promoter (eg the Saccharomyces cerevisiae PRB1 promoter), astaught in EP 431 880; a sequence encoding a secretion leader which, forexample, comprises most of the natural HSA secretion leader, plus asmall portion of the S. cerevisiae α-mating factor secretion leader astaught in WO 90/01.063; the HSA coding sequence, obtainable by knownmethods for isolating cDNA corresponding to human genes, and alsodisclosed in, for example, EP 73 646 and EP 286 424; and a transcriptionterminator, preferably the terminator from Saccharomyces ADH1, as taughtin EP 60 057. Preferably, the vector incorporates at least twotranslation stop codons.

The choice of various elements of the plasmid described above is notthought to be directly relevant to the purity of the albumin productobtained, although the elements may contribute to an improved yield ofproduct. A preferred embodiment of the fermentation and purificationprocess is described in Example 1.

Example 1

The cloning strategy for construction of the albumin-producingmicro-organism was as disclosed in EP 431 880 except that the 3′ end ofthe albumin coding sequences and its junction with the ADH1transcription termination sequence were altered such that the ADH codingsequence was eliminated and such that two consecutive in-frametranslation stop codons were present, followed by a third stop codondownstream, as follows:

SEQ ID NO. 9 . . . L    G    L    stop stop A    stop . . .TTA  GGC  TTA  TAA  TAA  GCT  TAA . . .

This was achieved by modification of the ADH1 terminator from plasmidpAYE309, described in EP 431 880, by PCR mutagenesis using two singlestranded oligonucleotides, JMADH1 and JMADH2 with the sequences:

JMADH1 SEQ ID NO. 10      HindIII 5′-GCATAAGCTTTGGACTTCTTCGCCAGAGGTTTGGTCAAG-3′ JMADH2 SEQ ID NO. 11                                  NotI    BamHI3′-TGGACAACATTAGCAAGAAGGTGTGCCTAG CGCCGGCGC CTAGGTA CG-5′

The PCR conditions were 25 cycles of 94° C. for 60 seconds, 37° C. for120 seconds and 72° C. for 180 seconds. The 0.48 kb PCR product wasdigested with both HindIII and BamHI and ligated into plasmid pBST+,described in WO 97/24445, similarly digested with HindIII and BamHI, tocreate plasmid pAYE440 (FIG. 2). The ADH1 terminator was furthermodified by PCR mutagenesis using two single stranded oligonucleotides,AT19R and the universal −40 primer with the sequences:

AT19R SEQ ID NO. 12            HindIII 5′-AGTCCAAGCTTAATTCTTATGATTTATGAT-3′ −40 SEQ ID NO. 13 3′-CAGCACTGACCCTTTTG-5′.

The PCR conditions were 25 cycles of 94° C. for 30 seconds, 50° C. for40 seconds and 72° C. for 50 seconds and then one cycle of 72° C. for 10minutes, using the ADH1 terminator in pAYE440 as a template (FIG. 2).The machine used was a Perkin Elmer GeneAmp PCR System 9600. A productof the correct size, approximately 0.33 kb, was obtained and digestedwith both HindIII and BamHI. Plasmid pAYE309, described in EP 431 880,was digested with NotI and HindIII and the 0.84 kb DNA fragmentcontaining the PRBI promoter fragment and part of the HSA/MFα-1 leadersequence (WO 90/01063) employed to direct secretion of mature HSA, wasligated into NotI and HindIII digested pBST+, described in WO 97/24445,to generate plasmid pAYE438 (FIG. 3). The recipient plasmid pAYE438 wasdigested with HindIII and BamHI and the modified ADH1 terminator wassuccessfully cloned into this vector to generate plasmid pDB2241 (FIG.4). This plasmid contains the pBST+ (WO 97/24445) backbone, the PAB1promoter and the modified ADH1 terminator.

To facilitate the introduction of two translation stop codons at the endof the HSA coding region and create the required HindIII site, the 3Nend of the HSA coding region was altered.

The double stranded oligonucleotide linker, AT21/AT22 was ligated intoAflII/HindIII cut pDB2241 and comprised an AflII site at its 5N end, astuffer region and then the Bsu36I to HindIII sequence of the HSA codingDNA, but with the addition of an extra DNA translation stop codon.Clones with the linker inserted were checked by DNA sequencing and thecorrect plasmid designated pDB2242 (FIG. 5).

Linker AT21/22 AT21 SEQ ID NO. 14AflII              Bsu36I              HindIII                                               SEQ ID NO. 15  TTA AGAGTC CAA GCC TTA GGC TTA TAA TA    CT CAG GTT CGG AAT CCG AAT ATT ATTCGASEQ ID NO. 16 A L G L Stop Stop

To create the final rHA expression cassette the AflII/Bsu361 fragment ofpAYE309 (FIG. 1) was ligated into AflII/Bsu36I digested pDB2242, makingplasmid pDB2243 (FIG. 6). Finally, the rHA expression disintegrationvector was made by ligating the NotI expression cassette from pDB2243into NotI cut pSAC35 (Sleep et al, 1991, Bio/Technology 9, 183-187 andEP 431 880) to generate the plasmid pDB2244 (FIG. 7) in which thedirection of rHA transcription is in the same orientation as that of theLEU2 gene.

The plasmid pDB2244 is therefore derived from the disintegration vectorpSAC3 (Chinery and Hinchliffe (1989) Current Genetics 16, 21-25) andcomprises the whole of the 2 μm plasmid, the LEU2 gene to complement thehost leu2 mutations, the expression cassette in which the PRB1 promoterdrives expression of the HSA sequence and the bacterial plasmid pUC9.The latter is excised from the plasmid by the S. cerevisiae 2 μm FLPrecombinase system such that no bacterial DNA is present in the organismused for production of rHA (Chinery and Hinchliffe, op cit.).

The expression vector utilises the S. cerevisiae PRB1 promoter and ADH1transcription terminator to control expression and the HSA/MFα-1 leadersequence (WO 90/01063) to direct secretion of mature HSA.

The plasmid pDB2244 was introduced into a Saccharomyces cerevisiaestrain which was leu2, yap3, hsp150, pmt1 [cir°] by the method describedby Hinnen et al, (1978) P.N.A.S. 75, 1929. The pmt1 mutation may beachieved by the method of WO 94/04687. Transformants were selected on abuffered minimal medium (0.15% (w/v) yeast nitrogen base without aminoacids and ammonium sulphate (Difco), 0.5% (w/v) ammonium sulphate, 0.1 Mcitric acid/Na₂HPO₄. 12H₂O pH6.5, 2% (w/v) sucrose)) lacking leucine.When transformants were grown for 72 hours at 30° C., 200 rpm in 50 mlflasks containing either 10 ml of complex (YEP, 1% (w/v) yeast extract,2% (w/v) bactopeptone and 2% (w/v) sucrose), or buffered minimal mediumliquid medium, rHA could be detected in the cell free culturesupernatant by SDS-polyacrylamide gel electrophoresis and/or by rocketgel immunoelectrophoresis.

A stock master cell culture in buffered minimal medium is used toprepare running stocks (working cell bank) of process yeast suitable forthe preparation of shake flask cultures by freezing aliquots of theculture in the presence of 20% (w/v) trehalose.

The fermentation was essentially the same as is described in WO 96/37515and U.S. Pat. No. 5,728,553, both of which are incorporated herein byreference, except for the following differences:

Seed Fermentation

After the medium for rHA production has been added to the seed fermentervessel, the operating temperature of 30° C. is set, as well as theminimum stirrer speed set to achieve homogeneity and so avoid gradientsof nutrients such as oxygen or carbon. The initial pH is adjusted withammonia solution (specific gravity 0.901) using a pH controller set at6.40; controlled at 6.40±0.10.

Alternatively, pH is maintained in the range of 5.50 to 5.90, with thelower control set point being 5.50. The initial pH may be adjusted withammonia (eg aqueous ammonia specific gravity 0.880). This lowerfermentation pH results in an enhanced mass spectrometry profile of therHA.

It is preferable for the initial pH to be near the top of theaforementioned ranges to facilitate observation of early metabolism,since a decline in pH is the first sign of growth detectable by on-lineinstruments.

Particularly for strains with a deficiency in one or more of the PMTgenes, it has been found to be beneficial for the fermentation to beconducted at a higher pH than is normally required. Thus, rather thancontrol the pH at approximately 5.5, it is beneficial to have a controlset point between pH6.20 and pH6.70, preferably between pH6.3 and 6.5.At such a higher pH, the quality of the centrate is significantlyimproved due to reduced cell lysis. Cell lysis results in cell debrisremaining in suspension following a centrifugation step of thefermentation which is sufficient only to remove all whole cells from thesupernatant. This is demonstrated in Table 1, where a significantreduction in the wet weight content of a culture supernatant is shownwhen the yeast is cultured in the pH range 6.3 to 6.5 compared to pH5.5.

TABLE 1 Relationship between centrate quality and fermentation pH inseed fermenter vessel. Wet Weight Content of Fermentation pH Supernatant(g · L⁻¹) 5.5 9.9 (2.4, 6)  6.3-6.5 3.4 (1.0, 13) Values in parenthesesare standard deviation and number of samples.

2M H₂SO₄ is also used as a pH corrective agent. Sucrose to 20 g·L⁻¹,MW10 batch vitamins, and Breox FMT30 antifoam to 0.04 g·L⁻¹ are added tothe vessel.

Sterile filtered air is introduced into the vessel at 0.5 v/v/m (ie 0.5litre non-compressed air per litre of medium per minute), the medium isinoculated to >10 mg cell dry weight L⁻¹ from axenic shake flask cultureand a supervisory computer control system is initiated. The expectedbatch phase is 62±10 h from an inoculum concentration of 12 mg·L⁻¹. MW10feed must be connected before the end of the batch phase (volume equalto batch volume).

Features of the fermentation control algorithm include: the end of batchphase being signalled by dissolved oxygen tension (DOT) increase of >15%in 30 min; the feed being initiated at 0.05 ml per litre batch medium;the substrate feed rate being determined according to the formula,SF=SF_(o)ek^(μk), wherein SF is substrate feed rate (mL·min⁻¹); SF₀ isinitial substrate feed rate (mL·min⁻¹), μ is specific growth rate (forexample 0.06 h⁻¹), and k is a counter variable started at 0 andincreased by 0.0167 once every 1 min if all conditions are met.; and thesubstrate feed rate (via manipulation of k) being reduced in response toDOT<15% and/or respiratory quotient (RQ)≧1.2.

The feed is stopped if the pH<6.2 or if the temperature<29.0° C.or >31.0° C. This may also be done automatically through the controlalgorithm. The SF is no reduced if the average RQ>1.13 over a 2 hperiod, or if there is evidence of ethanol or acetate accumulation.

Agitation is increased to maintain DOT>20% air saturation. Once the feedis started, the concentration of Breox FMT30 is increased to 0.3 g·L⁻¹(calculated on final volume). The expected feed phase duration is 65±17h, dependent upon transfer limitations of the vessel.

The air flow is increased through the fermentation to maintain thevalues of oxygen uptake rate (OUR) and carbon dioxide evolution rate(CER), at levels sufficient to provide accurate gas analysis. The airflow rate of the fermentation is nominally 1 v/v/m, Daily checks areperformed to determine purity of culture and CDW. Appropriate samplesare retained. At the end of the feed, the culture is transferred to aproduction vessel.

Production Fermentation

The production fermenter is inoculated at 0.25-1.00 g·cdw·L⁻¹. Theinitial pH is adjusted with ammonia solution (SG 0.901) using a pHcontroller set at pH6.40; so controlled at 6.40±0.10.

Alternatively, pH is maintained in the range of 5.50 to 5.90, with thelower control set point being 5.50. The initial pH may be adjusted withammonia (eg aqueous ammonia specific gravity 0.880). This lowerfermenation pH results in an enhanced mass spectrometry profile of therHA.

It is preferable for the initial pH to be near the top of theaforementioned ranges to facilitate observation of early metabolism,since a decline in pH is the first sign of growth detectable by on-lineinstruments.

Particularly for strains with a deficiency in one or more of the PMTgenes, it has been found to be beneficial for the fermentation to beconducted at a higher pH than is normally required. Thus, rather thancontrol the pH at approximately 5.5, it is beneficial to have a controlset point between pH6.20 and pH6.70, preferably between pH6.3 and 6.5.At such a higher pH, the quality of the centrate is significantlyimproved due to reduced cell lysis. Cell lysis results in cell debrisremaining in suspension following a centrifugation step of thefermentation which is sufficient only to remove all whole cells from thesupernatant. This is demonstrated in Table 2, where a significantreduction in the wet weight content of a culture supernatant is shownwhen the yeast is cultured at pH 6.5 compared to pH 5.5.

TABLE 2 Relationship between centrate quality and fermentation pH inproduction vessel. Wet Weight Content of Fermentation pH Supernatant (g· L⁻¹) 5.5 36.3 6.5 4.7

2M H₂SO₄ is also used as a pH corrective agent. Sucrose to 20 g·L⁻¹,MW10 batch vitamins, and Breox FMT30 antifoam to 0.04 g·L⁻¹ are added tothe vessel.

The initial substrate feed rate is determined according to the formula:

${SF}_{o} = \frac{1000 \times \mu \times \lbrack{CDW}\rbrack \times V_{batch}}{60 \times Y_{x/s} \times \lbrack{sucrose}\rbrack}$

wherein SF₀ is initial substrate feed rate, μ is specific growth rate(for example 0.06 h⁻¹), V_(batch) is batch volume (L), Y_(x/s) is cellyield (g·L⁻¹), [sucrose] is sucrose concentration (g·L⁻¹) and [CDW] iscell dry weight concentration (g·L⁻¹). The substrate feed rate isdetermined according to the formula, SF₀e^(μk), wherein SF is substratefeed rate (mL·min⁻¹); SF₀ is initial substrate feed rate (mL·min⁻¹), μis specific growth rate (h⁻¹) (for example 0.06 h⁻¹), and k is a countervariable started at 0 and increased by 0.0167 once every 1 min if allconditions are met. A number of conditions are constantly reviewedduring the fermentation, and used to adjust SF via manipulation of k; SFis reduced in response to DOT<15% and/or respiratory quotient (RQ)≧1.2.The feed is stopped if the pH<6.2 or if the temperature<29.0° C.or >31.0° C. This may also be done automatically through the controlalgorithm. The SF is reduced if the average RQ>1.13 over a 2 h period,or if there is evidence of ethanol or acetate accumulation.

Agitation increased to maintain DOT≧20% air saturation, and maintainedat a maximum once attained in order to facilitate mixing. Once the feedis started and the culture is under carbon limitation, the concentrationof Breox FMT30 is increased to 0.2-0.32 g·L⁻¹ (calculated on finalvolume). The expected feed phase duration is dependant upon transferlimitations of the vessel, typically 90-120 h at the 8,000 L scale.

The air flow is increased incrementally through the fermentation tomaintain the values of oxygen uptake rate (OUR) and carbon dioxideevolution rate (CER), at levels sufficient to provide accurate gasanalysis. The vessel is overpressured as necessary to enhance OTR. Theair flow rate of the fermentation is nominally 1 v/v/m. Daily checks canbe performed to determine purity of culture and CDW, and appropriatesamples are retained.

The culture is held for downstream processing at the end of the feed.

Hold of Production Culture

The production culture may be held under appropriate conditions toenable batch processing of the culture. The hold time should be kept toa minimum, but can be extended up to 48 hours and beyond if necessary(eg to 5 days). It will be appreciated that, under conditions of batchprocessing, the constraints of hold time as expressed herein apply tothe final portion of the culture to be processed.

The centrate from the fermentation, or an impure albumin solution fromany other source (such as plasma), is prepared, or conditioned, forchromatography on a cation exchange matrix while protecting the rHA frompolymerisation and protease activity. Preferably, sodium octanoate isadded (Chromatography Solution 14 (CS14)—Table 3) to a finalconcentration of 1-10 mM, for example approximately 5 mM. The pH isadjusted with acetic acid to pH4.3-4.8, preferably 4.50±0.1 (mostpreferably ±0.05) and the conductivity is checked to be <5.5 mScm⁻¹.

Chromatograhy

All operations can be carried out at ambient temperature (20±5° C.). Thealbumin loads (g/L) for the chromatography columns are determined fromtitres of albumin (g/L) by either SDS-PAGE (at the first step) orGP-HPLC (for all other columns). The progress of each step is monitoredby measuring UV absorbance on line, for example at 254 or 280 nm.

In a particularly preferred embodiment of the present invention thepurification process comprises the following steps: cation exchangechromatography (SP-FF); anion exchange chromatography (DE-FF); affinitychromatography (DBA); ultrafiltration and diafiltration; a secondaffinity chromatography step (PBA); ultrafiltration and diafiltration; asecond cation exchange chromatography step (SP-FF2); and a second anionexchange chromatography step (DE-FF2). Preferably, these purificationprocesses are followed by final ultrafiltration/diafiltration followedby a formulation step, and/or placing of the solution into a finalcontainer.

The sequence of chromatographic steps as described here is novel andinventive in a number of aspects. The use of an aminophenylboronate(PBA) matrix with an improved buffer, as described herein, and a smallload volume has been shown to give increased yeast antigen clearance, asmeasured by ELISA (about 4-20 fold). The buffer used with theaminophenylboronate matrix was unexpectedly found to be particularlybeneficial, and it represents the result of intensive trials of aplethora of buffers of widely ranging constituents and properties. Thebuffer provides a significantly increased clearance of yeast antigens,when compared with the buffer used in the PBA chromatography step of WO96/37515.

Loading the aminophenylboronate matrix with a highly concentratedalbumin solution, for example 10±10 g·L⁻¹, means that improvedresolution of the rHA and yeast antigens can be achieved because of thesmaller load volume.

WO 96/37515 includes a S200 gel permeation step after a first affinitychromatography step. The gel filtration step purified the albumin withrespect to yeast antigens, pigment and dimerised albumin. We have foundthat this step is no longer necessary because of the improvements wehave made to the aminophenylboronate affinity step and the introductionof additional cation and anion exchange steps.

Following the aminophenylboronate affinity step it is preferred that thealbumin is concentrated and diafiltered for a negative mode cationexchange step. We have found that the combination of this diafiltrationstep and cation exchange step substantially reduces the relativeconcentration of nickel ions. In particular, exposing rHA to a low pH iseffective in reducing nickel ion levels. Consequently, albumin purifiedaccording to the present invention has a surprisingly low nickel ioncontent (less than 100 ng/g of albumin).

The negative mode cation exchange step, as described herein, is used toremove Concanavalin A binding material (cbm) which is a small amount ofmodified rHA, thought to be glycosylated. The negative mode cationexchange step has been found to reduce the cbm content produced byrecombinant pmt1-mutant Saccharomyces cerevisiae by approximately1.3-fold. A greater effect is achieved with rHA derived from non-pmt1mutants (2-3 fold clearance).

In comparison with other commercial yeasts, Saccharomyces cerevisiaeproduces a relatively low level of modified rHA. Accordingly, thenegative mode cation exchange step and the use of cells with adeficiency in one or more of the PMT genes may be of even greaterimportance if the rHA is produced by a recombinant host other thanSaccharomyces cerevisiae.

The chromatography solutions used during the purification of albumin aredetailed in Table 3. Because of the very large scale manufacture ofalbumin, and the relatively low cost of the product, these buffer saltsare the most suitable for the process as they are available in a highlypure form at industrial scale and are low cost compared to othercommonly used buffers such as Tris, HEPES or MOPS. Alternative bufferscould be used in place of the ones used in Table 3, for example buffersof a similar pK_(a) (eg malate for acetate), but in most instances costand availability at large scale rule out their use. Alternative saltforms can be used provided they are soluble, available at industrialscale and low cost.

Chromatography can be performed using either axial flow columns, such asthose available from Pharmacia, or using radial flow columns, such asthose available from Sepragen. A fluidised bed may be used, at least forthe first step.

The buffer solutions can be prepared at the concentrations describedbelow, or concentrated stock solutions can be prepared and mixed ordiluted on-line for immediate use.

Cation Exchange Chromatography

Albumin is concentrated and purified with respect to at least yeastproteins (if the albumin is rHA from a yeast fermentation) and otherantigens, low molecular weight contaminants and pigmented compounds bycation exchange chromatography. The method uses a commercial cationexchange matrix such SP-Sepharose FF, SP-Spherosil, CM-Sepharose FF,CM-Cellulose, SE-Cellulose or S-Spherodex. Preferably, the matrix isSP-Sepharose FF (Pharmacia) which, if used in an axial flow column, maybe at a bed height of 5 to 25 cm, preferably 10 to 15 cm, for example12.5 cm. If a radial flow-type column is used, a suitable bed flow pathlength is 11.0±1.0 cm. A column loading of 10 to 50 g albumin/L,preferably 40±10 g albumin/L, of matrix is suitable. The matrix isequilibrated with a buffer to remove the alkali storage solution;preferably the buffer should be strong enough to reduce the pH toapproximately pH6.0. A buffer such as CS01 is used to remove storagesolution CS07 from the column; however, any buffer with a pH<6.0 couldbe used. Equilibration is Judged to be complete when the pH of thecolumn effluent is approximately pH6.0.

TABLE 3 Chromatography solutions for the purification of albuminSolution Concentration Conductivity No. Name Constituent (g · L⁻¹) pH(mS · cm⁻¹) CS01 SP-FF CH₃COOH 1.85 5.45-5.65 1.9-2.2 Equilibrant/Wash3/ NaOH (27% 4.00 DE-FF Equilibrant (w/w)) CS02 SP-FF Wash 1 CH₃COOH3.00 3.9-4.1 0.6-0.8 NaOH (27% 1.19 (w/w)) CS03 SP-FF Wash 2 CH₃COOH1.62 3.9-4.1 125-165 NaOH (27% 1.19 (w/w)) NaCl 117 CS04 SP-FF Eluent/CH₃COOH 5.13 5.4-5.6 5.0-6.0 DE-FF Pre- NaOH (27% 11.5 Equilibrant(w/w)) Octanoic Acid 0.721 CS05 Salt Clean NaCl 58.4 5-9 75-95Polysorbate 80 5.00 CS06 0.5M NaOH NaOH (27% 74.1 >12  80-120 (w/w))CS07 20 mM NaOH NaOH (27% 2.96 >12 3.5-5.5 (w/w)) CS08 DE-FF WashK₂B₄O₇•4H₂O 4.80 9.0-9.4 2.5-3.5 CS09 DE-FF Eluent K₂B₄O₇•4H₂O 33.69.2-9.5 15.0-18.0 CS10 DBA CH₃COONH₄ 19.3 8.7-9.1 18-22 Equilibrant/WashNaOH (27% 5.93 (w/w)) CS11 DBA Eluent NaCl 117 6.7-7.1 125-165 NaOH (27%14.1 (w/w)) H₃PO₄ 5.79 (85%(w/w)) CS14 2M Sodium NaOH (27% 281 7.8-8.4 —Octanoate (w/w)) Octanoic Acid 288 CS15 Acetic Acid CH₃COOH 1045 — —CS17 DE-FF2 CH₃COOH 1.50 4.5-4.7 0.85-1.05 Equilibration/Wash NaOH (27%w/w) 1.66 CS18 Positive-mode NaH₂PO₄•2H2O 8.58 6.8-7.0 5.5-6.5 DE-FF2Elution NaOH (27% w/w) 4.07 CS19 SP-FF2 CH₃COOH 1.80 5.2-5.4 1.8-2.1Equilibration/Wash NaOH (27% w/w) 3.52 CS20 PBA Glycine 7.51 8.3-8.618-22 Equilibration/Wash NaCI 5.84 NaOH (27% w/w) 0.95 CaCl₂•2H₂O 7.35CS21 20%(w/w) Acetic CH₃COOH 205 1.9-2.2 1.8-2.0 Acid H₂O 820 CS22 FinalpH Na₂HPO₄ 71.0 11.2-11.4 43-49 Adjustment NaOH (27% w/w) 37.0 EXO4Final pH adjustment NaOH (47% w/w) 42.6 ≧12  80-120 alkali H₂O 970 EXO5Final pH adjustment HCl (37% w/w) 19.7 ≦1.5 60-90 acid H₂O 982 Allweighings are ±2%, for this particular example.

The centrate from a fermentation is prepared, or conditioned, forchromatography on a cation exchange matrix while protecting the rHA frompolymerisation and protease activity. However if the yeast strain is notdeficient in the proteases that degrade rHA at the pH required to purifythe rHA then the culture supernatant should be pasteurised, for exampleby a heat treatment of 50-70° C. for 30 minutes to 5 hours, as detailedin WO 94/03636. Typically 1-10 mM sodium octanoate is sufficient toprotect the rHA from heat denaturation and 30 seconds up to 10 minutesat temperatures of 60-80° C. adequate to inactivate the proteases in abatch or flowthrough procedure. Pasteurisation may also be desirable ifHSA is used.

The conditioned centrate is then loaded onto the column at a flow rateof, for example, 0.07-0.75 bed volumes/min, preferably 0.3-0.6 bedvolumes/min, in this example 0.5 bed volumes/min, and then the column iswashed with one or more solutions to remove residual contaminants. Thecolumn is washed first with, for instance, eight volumes of 10-100 mM,preferably 30-70 mM, for example 50 mM acetate, pH3.9-4.1, 0.6-0.8mS·cm⁻¹ (CS02). The column is then washed with four volumes of a highsalt buffer containing 1-3M NaCl, preferably 2M NaCl, in sodium acetatebuffer (for example 10-50 mM sodium acetate, preferably about 27 mM,pH3.5-4.5, preferably pH4.0 (CS03) and then ten volumes of CS01. Thealbumin is eluted with, and collected in an acetate/octanoate buffer(for example 40-120, preferably 60-100, eg 85 mM acetate, and 2-50 mM,preferably, 2-20 mM, eg 5 mM octanoate, as in CS04). The collection ofalbumin starts when the UV signal rises above 0.6 A₂₅₄/cm, andcollection continues until the UV signal Falls below 0.36 A₂₅₄/cm. Thecolumn is then cleaned using 0.25-3.0M NaCl and 0.05-2% detergent (CS05)and then 0.1-1.0M NaOH(CS06), then stored in diluted (10-50 mM) NaOH(CS07), In this example, the flow rate for the equilibration, loadingand wash steps is 0.5 bed volumes per minute. For elution of thealbumin, a flow rate of 0.04-0.6 bed vol/min, preferably 0.15-0.35, inthis example 0.25 bed vol/min is used.

Anion Exchange Chromatography

The eluate from the cation exchanger is then diluted to below 10mS·cm⁻¹, preferably less than 5 mS·cm⁻¹, especially below 2.5 mS·cm⁻¹and then loaded onto an anion exchange resin such as QMA-Spherosil,DEAE-Spherodex, Q-Hyper D, DEAE-cellulose, QAE-cellulose, or TMAE, DMAE,or DEAE Fractogel. Preferably, the matrix is the commercial anionexchange matrix DEAE Sepharose-FF (Pharmacia), bed flow path length of11.0±1.0 cm, pre-equilibrated with the cation elution buffer (CS04) andthen equilibrated with three column volumes of CS01. The albumin isloaded onto the matrix at 30±10 g monomeric albumin per litre of matrixand then the matrix is washed with dilute tetraborate buffer, forexample 15-25 mM potassium tetraborate or sodium tetraborate (CS08),which has the effect of raising the pH to approximately 9.2, and thenthe albumin is eluted with a more concentrated tetraborate buffer (forexample 80-150 mM potassium tetraborate, preferably 110 mM potassiumtetraborate (CS09)). The matrix is cleaned with salt/detergent (CS05)and then NaOH(CS06) before storage in dilute NaOH (CS07). The eluatefrom the anion exchange matrix is then loaded onto an affinity matrix.

Affinity Chromatography

This step further purifies the rHA with respect to a 45 kDa N-terminalalbumin fragment, yeast antigens and pigment. The affinity matrix maycomprise any Cibacron Blue type of dye which binds albumin, for exampleReactive Blue 2, Procion Blue HB, Blue Sepharose, Blue Trisacryl andother anthraquinone-type compounds. Preferably, the matrix is the “DeltaBlue”⁷ Matrix (DBA), prepared as described in WO 96/37515.

The method uses DBA at a bed flow path length of 11.0±1.0 cm. The DBA isequilibrated in ammonium acetate buffer (100-300 mM, preferably 200-275mM, for example 250 mM as in CS10) and the albumin applied at 7.0-14.0g/L, preferably 8.0-12.0 g/L, in this example 10.0±1.0 g/L.Equilibration, load and wash steps are performed at flow rates of0.05-0.30 bed vol/min, preferably 0.15-0.27, in this example 0.25 bedvol/min. All other steps are performed at 0.20 bed vol/min. When loadingis complete, the column is washed to remove contaminants with 1-5volumes of ammonium acetate buffer 10-30 mS cm⁻¹, preferably 15-25 mScm⁻¹, for example CS10, preferably 5 column volumes. The albumin iseluted with a strong salt and phosphate solution (1.0-3.0M NaCl, forexample 1.5-2.5M NaCl or 2.0M NaCl, and 5-100 mM, eg 50 mM phosphate, asin CS11. The column is then cleaned using CS06 and stored in CS07.

The eluate from the DBA column is then concentrated and diafiltered inpreparation for purification using phenyl boronate agarose (PBA)chromatography. DBA ultrafiltration can be performed with anyultrafiltration membrane used in protein concentration with a nominalmolecular weight cut off of 30,000 or less, preferably apolyethersulphone type membrane (eg Filtron Omega series) of 10,000nominal molecular weight cut off. DBA eluate is concentrated and thendiafiltered at ≈100 g rHA·L⁻¹ against at least 5 volumes of waterfollowed by at least 5 volumes of CS20. At the end of diafiltration, theretentate may be further concentrated if required and the equipmentwashed out with CS20 to increase step recovery. The concentration of thefinal retentate should be in the range 20-120 g rHA·L⁻¹, preferably70-120 g·L⁻¹, or as in this example 100±10 g rHA·L⁻¹. After use, themembranes are treated by flushing out residual protein with water,cleaning with CS06 and storage in CS07.

PBA is an affinity step to remove glycoconjugates, such asglycoproteins, glycolipids and poly-, oligo- and monosaccharides, andutilises immobilised aminophenylboronic acid as the ligand. Theaminophenylboronic acid is covalently coupled via a spacer to aninsoluble matrix such as polyacrylamide, agarose, cellulosic or organicpolymers. U.S. Pat. No. 4,562,251 (incorporated herein by reference)describes suitable methods for making diborotriazine or monoborotriazineagarose: (1) triazine is O-linked to agarose first and then linked with3-aminophenylboronic acid (APBA) in a second reaction. (2) Triazine isreacted with APBA first to produce either mono or diborotriazine. Theseare then O-linked via the free chlorine on the triazine to the —ONaactivated agarose to produce either mono or disubstituted agarose.

An earlier patent, U.S. Pat. No. 4,269,605, contemplates a variety ofactivation methods, including epichlorohydrin activation of agarose,preferred herein. Commercially available matrices include Amicon's PBA30and Sigma's acrylic beaded aminophenylboronate.

It has been found to be particularly beneficial to use a buffercontaining glycine (10-500 mM, for example 25-200 mM, preferably 50-150mM, in this example 100 mM), NaCl (0-500 mM, for example 25-200 mM,preferably 50-150 mM, in this example 100 mM) and CaCl₂ (5-250 mM,preferably 10-100 mM, in this example 50 mM), pH8.0-9.5, preferably, pH8.0-9.0, in this example pH8.5 (CS20).

The PBA column uses a flow path length of 11.0±1.0 cm and ispre-equilibrated with the buffer as described above, eg CS20. The columnis loaded at less than 1 column volume, preferably less than 0.5 columnvolumes, in this example ≦0.35 column volumes. The PBA is ran as anegative step and therefore the albumin is collected in the flow throughand wash from the column. All chromatographic steps can be performed atflow rates of 0.005-0.3 bed vol./min.

Preferably the equilibration and cleaning of the column are carried outat a higher flow rate, eg 0.19 bed vol./min, than the load andcollection of the albumin solution, which is preferably carried out at aflow rate of 0.01-0.05, preferably 0.025 bed vol./min. The column isthen cleaned with salt (CS03), borate buffer (CS09), NaOH(CS06) and thenstored in dilute NaOH(CS07).

Following PBA chromatography the albumin solution is concentrated anddiafiltered to prepare for a negative mode cation exchange step. Thecombination of this diafiltration step and the negative mode cationexchange chromatography substantially reduces the relative concentrationof nickel ions.

PBA ultrafiltration can be performed with any ultrafiltration membraneused in protein concentration with a nominal molecular weight cut off of30,000 or less, preferably a polyethersulphone type membrane (eg FiltronOmega series) of 10,000 nominal molecular weight cut off. The collectedPBA Flow Through is adjusted to pH5.3±0.5 with CS21, concentrated andthen diafiltered at ≈100 g rHA·L⁻¹ against at least 7 volumes of CS19.At the end of diafiltration, the equipment is washed out with CS19 andfurther CS19 added as required to give a retentate concentration of50±10 g rHA·L⁻¹. Finally, sodium octanoate is added to give a finalconcentration of approximately 2-15 preferably 5-10, more preferably6-9, and in this example 6 mM, eg CS14 is added to 3 mL·L⁻¹. After use,the membranes are treated by flushing out residual protein with water,cleaning with CS06 and storage in CS07.

The albumin solution is then subjected to a second cation exchange stepusing, for instance, SP-FF Sepharose (Pharmacia), this time in thenegative mode, ie the albumin passes through the matrix, rather thanbeing retained. The conditions are such that mannosylated albumin bindsto the matrix. The buffer is preferably a sodium acetate buffer (5-110mM acetate, preferably 10-50 mM, in this example 30 mM), pH 5.2-5.4,CS19). Other buffers which can buffer in the appropriate range may beused, such as a citrate phosphate buffer. Suitably, the buffer has aconductivity of about 2 mS·cm⁻¹. The column has a flow path length of11.0±1.0 cm, with the albumin loaded to 10-250 g·L⁻¹ preferably 20-70g·L⁻¹ and in this example 50±15 g or 50±10 g·L⁻¹ matrix. Since this is anegative step, the albumin is collected in the flow through and wash.

Following this cation exchange step, the albumin is subject to negativemode anion exchange chromatography. This step removes yeast antigens asmeasured by ELISA and Western blot. The collected flow through and washfrom the second cation exchange step is adjusted to pH4.60±0.10 withCS21, diluted to 1.05±0.1 mS·cm⁻¹ with water and the rHA purified usingthe following conditions. The step uses an anion exchange matrix such asDE-FF Sepharose (Pharmacia) at a flow path length of 11.0±1.0 cm and thealbumin is loaded to 50-250 g·L⁻¹, preferably 150±50 g·L⁻¹, matrix.Since this is a negative step, the albumin is collected in the flowthrough and wash. The pH of the Flow Through and Wash is then adjustedto 7.0±0.1 with CS22.

Alternatively, as described in Example 9, pH-adjustment may occur in theFinal UF feed vessel instead of being performed on the DEAE flow throughand wash.

While Example 1 has been illustrated with reference to a pmt1 mutant, itshould be appreciated that the purification process of the presentinvention is equally applicable to host cells which are not mutant atthis locus, or indeed which are not mutant at any other pmt locus

Example 2

Two assays were used to investigate centrate quality. The poorer thecentrate quality the worse the “robustness” of the yeast cells.

The two assays were:

1. Determination of the absorbance of centrate at 600 nm (A₆₀₀).2. Determination of the wet weight of particles in the centrate (WW).

In both the assays, the higher the value the poorer the centratequality.

The centrate quality of three different yeast strains tender twodifferent pH conditions grown in fed-batch fermentation were compared.

TABLE 4 A600 and WW values for three different rHA producing strains infed-batch fermentation grown at two different pH values. In the firstcolumn the specific gene deletions are indicated. Specific genedeletions A₆₀₀ WW (g · L⁻¹ centrate) Grown at pH 5.5 pmt1-/hsp150-/yap3-1.39 (0.52, 24) 12.4 (4.9, 23) hsp150-/yap3- 1.11 (0.62, 9)  9.1 (2.9,7) yap3- 0.58 (0.34, 10)  3.9 (2.0, 10) Grown at pH 6.4 or 6.5pmt1-/hsp150-/yap3- 0.41 (0.17, 6)  2.6 (0.8, 6) hsp150-/yap3- 0.47(0.19, 8)  4.6 (1.4, 7) yap3- 0.41 (0.08, 6)  2.1 (0.8, 6) Values inparentheses are standard deviation and number of samples.

From the Table above it can be concluded that at pH 5.5, themultiply-gene deleted strains yield an inferior centrate, whereas atpH6.4 or 6.5, the deleterious effect of these further gene deletions isavoided.

Example 3

This example was performed in the same manner described in Example 1,but utilised a strain which is not pmt1 mutant. This strain was alsogrown at two different pH control values, and the wet weight content ofthe centrate determined as described in Example 1. The benefit of growthat the elevated pH control point is also seen for this strain of yeast;demonstrated in Table 5, where a significant reduction in the wet weightcontent of a culture supernatant is shown when the yeast is cultured inthe pH range 6.3 to 6.5 compared to pH5.5.

TABLE 5 Relationship between centrate quality and fermentation pH fornon-pmt1 strain. Wet Weight Content of Fermentation pH Supernatant (g ·L⁻¹) 5.5 10.0 (2.3, 4) 6.3-6.5  4.6 (1.4, 7) Values in parentheses arestandard deviation and number of samples.

Thus, rather than control the pH at approximately 55, it is beneficialto have a control set point between pH6.20 and pH6.70, preferablybetween pH6.3 and 6.5. At such a higher pH, the quality of the centrateis significantly improved due to reduced cell lysis

Example 4

This Example was performed in a similar manner as described in Example1, with the following differences. The yeast Pichia pastoris, strainGS115 (Invitrogen) was grown using the same conditions and medium asdescribed above, but using a pH1 controller set at 5.90; controlled at5.90+0.20, a specific growth rate of 0.10 h⁻¹ with glucose as a carbonsource. The batch phase duration was 28 h, and the feed phase durationwas 42 h. Recombinant human albumin was added once the feed phase hadcommenced, providing a final concentration of 3.8 g rHA·L⁻¹ culture atthe end of the fermentation. The rHA used to spike the Pichia culturehad been purified but not in accordance with the purification process ofthe invention.

The rHA from the Pichia fed-batch culture medium was then purified inaccordance with the purification process described in Example 1.

Example 5

This Example describes the analysis of rHA purified from Pichia culturemedia as described in Example 4.

Immunoassay Data

Immunoassays were performed on: (i) the rHA purified from the Pichiaculture media; (ii) the rHA used to spike the culture media; and (iii)on albumin produced by Saccharomyces cerevisiae which had purified inaccordance with the present invention.

Western Blot Summary

Antibody Batch Number Ig9601 Gel Type 4-12% SDSNR NOVEX GELS Milk TypeUHT Exposure Time 20 seconds

Ig9601 was raised against a non-albumin producing Saccharomycescerevisiae strain and thus can be used to detect yeast antigens.

The western blot showed that the yeast antigen profile of the albuminderived from the Pichia culture medium contained fewer and less intensebands than the material used to spike the Pichia fermentation. ThePichia-derived albumin yeast antigen profile was very similar to theSaccharomyces-derived profile.

ELISA Blot Summary

Yeast antigen impurities in the albumin purified from the Pichia culturemedium and for the albumini used to spike the Pichia medium werequantified by ELISA using Ig9601.

The yeast antigen content of the albumin purified from the Pichiaculture medium was below the detectable limit of the assay(approximately 0.004 μg·g⁻¹), and the antigen content for the albuminused to spike the Pichia medium was 0.62 μg·g⁻¹.

Con A Binding Material

The Con A assay described in Example 9 was performed on albumin purifiedfrom the Pichia culture medium and for the albumin used to spike thePichia medium. The content of Con A binding material for the former was0.22% (w/w) and for the latter it was 0.57% (w/w).

The level of Con A binding material in the albumin purified from thePichia culture medium is similar to that of albumin purified fromSaccharomyces cerevisiae in accordance with the invention (see Table 6),when the latter is not produced from a pmt1 mutant.

The purity analyses confirm that the process of the invention can besuccessfully used to purify albumin from yeast other than Saccharomycescerevisiae (eg Pichia) and that albumin of similar purity to thatpurified from Saccharomyces cerevisiae can be obtained.

Example 6

In Example 1a negative mode anion exchange chromatography step (DE-FF2)followed the second cation exchange chromatography step (SP-FF2). In analternative purification process the second cation exchangechromatography step may be followed by a positive mode anion exchangechromatography step.

From the SP-FF2 eluate at pH5.3 approx. the pH needs to be increased topH7. There are two means detailed below, pH adjustment anddiafiltration. The latter appeared to give a better quality product.

DE-FF2 (A)

SP-FF2 flow through and washings were pH adjusted to pH 7.0 with 0.5 Mdisodium hydrogen orthophosphate. This material was loaded onto a DEAEunder standard positive conditions to give a matrix loading of 40 grHA·L⁻¹ matrix, the pH and conductivity of the load were 7.0 and 1.29mS·cm⁻¹ respectively.

DE-FF2(B)

SP-FF2 flow through and washings were diafiltered vs. 10 vol. 10 mMsodium phosphate pH 7.0, concentrated and diluted with buffer to 50g·L⁻¹ and loaded onto a DEAL under standard positive conditions. The pHand conductivity of the load was 7.0 and 1.43 mS·cm⁻¹ respectively. Thealbumin from DE-FF2A/DE-FF2B is suitably eluted by a 45-55 mM sodiumphosphate buffer (pH7.0).

Example 7

The kinetics of nickel removal from rHA by treatment with low pH wereinvestigated (see FIG. 9). The results showed that between pH 4 and 4.5both the rate and extent of nickel removal were independent of pH, butthat at pH 5 the rate of removal slowed slightly. Both the rate andextent of nickel removal decreased with increasing pH across the range5-6.5 with little or no removal above pH 6.5.

Example 8

Purification of human serum albumin from a sample of cryo-poor plasmapaste (Centeon Pharma GmbH) was achieved using the purification processdetailed in Example 1.

Recoveries of HSA at each chromatography step were predominantlycomparable to the anticipated rHA recovery at the same stage, with theexception of the PBA column. Here, the recoveries were much lower thanexpected which may have been due to removal of glycated albumin.

Example 9

This Example illustrates the concentration, diafiltration andformulation of the highly purified rHA into a suitable product, in thisinstance 20% (w/v) albumin. This procedure is carried out in two stages,namely final ultrafiltration (UF) and Formulation.

Final UF reduces nickel concentration by diafiltration at low pH andpresents rHA in a defined aqueous environment, using water of anappropriate grade,

Final UF begins with transfer of DEAE flow through and wash to the FinalUF feed vessel. As described below, the albumin is then concentrated,diafiltered pH adjusted to pH7.0 and further concentrated.

If DLE-FF2 is run in positive mode, the DE-FF2 eluate may be usedinstead of, or in addition to, the DEAL flow through and wash.

Following transfer of the DE-FF2 flow through and wash (or eluate ifDE-FF2 is run in positive mode), the rHA-containing process stream issequentially subjected to primary concentration, diafiltration andsecondary concentration phases in an ultrafiltration system fitted withcellulosic membranes with a nominal molecular weight cut off limit of10,000. The initial concentration step increases the rHA concentrationto approximately 100 g·L⁻¹ and is immediately followed by the continuousdiafiltration phase where the rHA is diafiltered against at least 5,preferably at least 7 retentate volume equivalents ofwater-for-injection, preferably a 50 mM salt solution to remove ammonia.Following diafiltration the pH is adjusted to 7.0 and, the secondaryconcentration phase further increases the rHA concentration to 275-325g·L⁻¹. At the end of UF the retentate is transferred to the bulk productformulation vessel.

Instead of pH-adjustment being performed on the DEAE flow through andwash, pH adjustment may occur in the Final UF feed vessel, preferablybetween the diafiltration process and the secondary concentration phase.Preferably, the diafiltration retentate is adjusted to pH 7±0.1 withEX04. If the pH exceeds 7.1 but remains <pH8.5 then the pH can bedecreased with EX05.

The formulation step produces rHA in an appropriate chemical environmentand at an appropriate concentration suitable for bulk: product sterilefiltration and filling. The transferred Final UF retentate is analysedto determine concentrations of albumin, sodium and octanoate. Thesequantities are taken into account and any necessary further amounts ofstock sodium chloride and sodium octanoate excipient solutions andappropriate grade water added to achieve the bulk formulationspecification. The final albumin concentration may be 150-250 g·L⁻¹ or235-265 g·L⁻¹ with a sodium concentration of 130-160 mM. Any otherfeasible albumin concentration may be made, however, with, for example,a minimum concentration of at least 4% (w/v), preferably 4-25% (w/v).Formulation is complete following addition of appropriate conventionalpharmaceutically acceptable excipients, such as polysorbate 80 or thosespecified in the US Pharmacopoeia for human albumin, and diluting water.

A final concentration of 0.08 mmoles sodium octanoate per gram ofalbumin may be desirable. The product is sterile and non-pyrogenic.There may be up to 1% dimeric albumin but no larger polymers oraggregates are detectable.

Example 10

This Example illustrates the analysis that is carried out to establishthe purity of albumin purified in accordance with the present invention.Unless stated otherwise, all of the assays are performed on albuminwhich has been purified according to Example 1 and formulated accordingto Example 9.

Glycation of rHA

A microassay for glycated protein has shown that rHA purified inaccordance with the invention is not substantially modified bynon-enzymic glycosylation (glycation). The microassay measures thestable Amadori product (AP) form of glycated protein, by oxidation ofthe C-1 hydroxyl groups of AP with periodate. The formaldehyde releasedby periodate oxidation is quantitated by conversion to a chromophore,diacetyldihydrolutidine (DDL), by reaction with acetylacetone inammonia. DDL is then detected calorimetrically. The samples were assayedafter desalting using a Pharmacia PD-10 (G25 Sephadex) column and thenthe albumin in the samples was re-quantitated by the Bradford method and10 mg albumin was assayed. A fructose positive control was included, andthe absorbances were read on a Shimadzu UV 2101 spectrophotometer at is412 nm. For every mole of hexose one mole of Amadori product is formed.

Moles Amadori Sample Product/Moles Albumin A 0.79 B 0.76 C 0.41 D 0.48 E0.46 F 0.22 G 0.41 H 0.37 I 0.54 J 0.76 K 0.84 L 0.50 M 0.43 N 0.59 O0.41 P 0.18 Q 0.24 R 0.04

Samples A-Q are commercially available HSA products from US, Europe andJapan (mean=0.49±0.20). Sample R is rHA purified according to theinvention.

Analysis of C-terminus

An important aspect of the quality control of recombinant proteins isthe conformation and stability of the pre-determined primary structure.Analysis of the C-terminal tryptic peptide in commercially available USAand rHA purified according to the invention by N-terminal sequencing andFAB mass spectometry indicated the presence of a truncated peptide,lacking the C-terminal leucine in HSA. The Des-Leu C-terminal trypticpeptide was detected in commercial HSA at approximately 5-10% (notquantitative), but could not be detected in the rHA of the invention,even after 6 months at 30° C. The Des-Leu peptide could not be detectedin the USA 12 weeks at 30° C., and the peak for the full lengthC-terminal peptide was very diminished compared to the other samples,indicating that perhaps this had undergone further C-terminaldegradation.

These results indicate that the rHA, purified in accordance with theinvention, has a stable and full length carboxy-terminus, whereas USApreviously available from commercial sources appears to be heterogeneousby comparison.

Nickel Ion Content of rHA Prepared According to the Invention MeasuringInstrument:

SIMAA 6000, Perkin Elmer Furnace: CTT (Constant Temperature Tube) usingdetection at 232 nm, 2470° C.

Calibration:

The method is based on a three-point calibration (18/30/60 lg/L standardsolutions from Perkin Elmer). After the calibration, a blank of purifiedwater is measured. The control standard is measured after the blank andat the end of each test series (Ni-Standard 20 lg/L, certified standardfrom Perkin Elmer).

Sample Preparation:

Each assay is the result of a determination in duplicate which alsovalid for the calibration and the control standard. Depending on theexpected Ni concentration, the sample is diluted in an appropriate ratioto work with a Ni-concentration that is within the calibration range.Samples with a protein concentration of 10% or more have to be dilutedat least 1:5 in any case. Dilution is with purified water.

Rinsing solution for the sample capillary: 2 L purified water mixed with0.5 mL Triton X100. Each test series includes a system suitability test.

Requirements:

1. Correlation coefficient of the calibration at least 0.99000. If not,the calibration has to be repeated one time. If the calibration does notcomply with the requirement a second time, an error analysis has to becarried out.2. Characteristic mass measured with the 30 lg/L-Standard may not exceedthe theoretical value of 20 pg/0.0044A-s by more than 20 percent.Characteristic Mass m₀:

That amount of the analyte in picogram (pg) that contributes anabsorption of 1 percent. An absorption of 1 percent corresponds to0.0044 A-s (ampere seconds).

$m_{0} = \frac{\begin{matrix}{{volume}\mspace{14mu} {Standard}\mspace{14mu} ({mL})*} \\{{{concentration}\mspace{14mu} \left( {{mg}\text{/}L} \right)*0.0044\; A} - s}\end{matrix}}{{absorption}\mspace{14mu} {sample}*{absorption}\mspace{14mu} {blank}}$

3. The measured concentration of the control standard has to be withinthe confidence range (2 s/3 s criterion).

Calculation:

The measuring instrument calculates the result according to thefollowing term:

${{Result}\mspace{14mu} \left( {g\mspace{11mu} {{Ni}/L}} \right)} = {\left( {\frac{A\; 1}{slope} \pm \frac{A\; 2}{slope}} \right)\text{:}\mspace{14mu} 2*V}$

A: absorptionslope: slope of the calibration curve (linear regression)V: dilution

A modifier is not used.

[Nickel]/[rHA] (μg/g) Sample Batch 1 Batch 2 PBA load 0.73 0.74 PBA flowthrough and wash 0.41 0.43 SP-FF2 load 0.06 0.06 SP-FF2 flow through andwash <0.03 <0.03 DE-FF2 flow through and wash 0.14 0.28

Analysis of Medium and Long Chain Fatty Acids

The fatty acids profiles of albumin according to the invention andcommercially available TISA were analysed by acidic solvent extractionand gas chromatography of the free fatty acids using a C17:0 internalstandard. No abnormal fatty acids have been detected in the albumin ofthe invention by this method although the profiles for the rHA and HSAshowed significant differences. As expected, both showed large amountsof the added stabiliser, octanoate (C8:0). Apart from this, commercialHSA was characterised by predominantly C16:0, C16:1, C18:0, C18:1 andC18:2 whilst the albumin of the invention contained mainly C10:0 andC12:0 and occasionally C14:0. Further experiments showed that the levelsof C10:0 and C12:0 in rHA final product correlated with the levels ofthese contaminants in the octanoate used for the latter stages of thepurification process.

Preferably, the total level of C18 fatty acids does not exceed 1.0%(mole/mole) of the level of octanoate, and preferably does not exceed0.5% of that level. Moreover, in the albumin of the invention, the levelof C18:2, C18:3 and C20 fatty acids is generally undetectable. Incommercial HSA, there may typically be about 0.4 mmoles C18 fatty acidsper mole of albumin. In the product of the invention, there aretypically no detectable C20 fatty acids and only about 0.02 moles C18fatty acids per mole of albumin.

SDS Reducing Polyacrylamide Gel Electrophoresis

This assay was performed as described in WO 96/37515. The assay showedthat rHA of the invention consists of a single polypeptide chain whichwhen treated with a reducing agent (β-mercaptoethanol) migrates as asingle band (monomer) on SDS reducing polyacrylamide electrophoresis(PAGE) which indicated that the proportion of albumin present as amonomer is at least 99.9%.

Gel Permeation High Pressure Liquid Chromatography

25

of a 10 mg/ml solution of albumin purified in accordance with theinvention which had been formulated to 25% w/v was injected onto aTSK3000SWXL column on a Shimiadzu LC6A HPLC and found to contain lessthan 0.1% polymeric albumin. This result indicates that the formulationas described herein has no detrimental effect on the polymer/aggregatecontent of the purified albumin.

Two Dimensional Gel Electrophoresis

2 μg rHA of albumin prepared by the process of the invention was subjectto two-dimensional electrophoresis using a Millipore Investigatorsystem. The separation in the first dimension was a pH 3-10 isoelectricfocusing gel and was followed by a 10% polyacrylamide/SDS gel in thesecond dimension. On staining of the gel with Coomassie Blue, only onespot was visible, indicating the presence of only one protein species.

Mannosylated Albumin/Con A Assay

Concanavalin A (Con A) binds molecules which contain α-D-mannopyranosyl,α-D-glucopyranosyl and sterically related residues. In the Con A assay,Con A Sepharose (Pharmacia, Cat. No, 17-0440-01) affinity chromatographyof recombinant Human Albumin (rHLA) and/or Human Serum Albumin (HSA) isused to determine the content of mannosylated albumin.

Recombinant human albumin (rHA) is diluted to 5% (w/v) rHA with 145 mMsodium chloride then 1:1 with Con A dilution buffer (200 mM sodiumacetate, 85 mM sodium chloride, 2 mM magnesium chloride, 2 mM manganesechloride, 2 mM calcium chloride pH5.5). 100 mg rHA is then loaded ontoan equilibrated 2 mL Con A Sepharose column which is then washed (5×4mL) with Con A equilibration buffer (100 mM sodium acetate, 100 mMsodium chloride, 1 mM magnesium chloride, 1 mM manganese chloride, 11 mMcalcium chloride pH5.5). The column is eluted with 6 mL Con A elutionbuffer (100 mM sodium acetate, 100 mM sodium chloride, 0.5Mmethlyl-α-D-mannopyranoside pH5.5).

Monomeric albumin in the Con A load (diluted to about 0.1 mg·mL⁻¹) andeluate (assayed neat) are quantified by GP.HPLC using a 0-0.2 mg·mL⁻¹rHA standard curve and the Con A binding albumin monomer recovered inthe eluate is expressed as a percentage of the load.

TABLE 6 Clearance of conA-binding rHA through the process. Batches 1-4are derived from a pmt1 mutant, whereas batch 5 is derived from anon-mutant strain. ConA-binding rHA (% of load) Batch 1 Batch 2 Batch 3Batch 4 Batch 5 PBA 0.14 PBA 0.16 PBA 0.15 PBA 0.13 PBA 0.55 FT&W FT&WFT&W FT&W FT&W SP-FF2 0.10 SP-FF2 0.12 SP-FF2 0.14 SP-FF2 0.09 SP-FF20.32 FT&W FT&W FT&W FT&W FT&W Final 0.10 Final 0.11 Final 0.12 Final0.07 Final 0.28 Product Product Product Product Product (FT&W = FlowThrough & Washings)

ConA-binding rHA was further analysed by electrospray mass spectrometry(FIG. 8). This indicated that, in addition to a reduction in the amountof conA-binding rHA, the extent of modification of the conA-binding rHAwas reduced.

Analysis of Colour

The absorbance of a 5% (w/v) solution of the final product in a 1 cmcuvette was measured at 350 nm, 403 nm and 500 nm and calculated interms of absorbances per gram of albumin/litre per cm pathlength (ieABS.L·g⁻¹·cm⁻¹). The albumin of the invention has the following values:

Mean absorbance Wavelength (n = 4 batches) (nm) (L · g⁻¹ · cm⁻¹) 3505.75 × 10⁻³  403 1.7 × 10⁻³ 500 0.4 × 10⁻³

Generally, the albumin of the invention does not exceed respectiveabsorbiances of 8.0×10⁻³, 3.0×10⁻³ and 0.75×10⁻³ at the said threewavelengths.

Assays of a number of commercially available HSA preparations revealedhigher absorbances at these wavelengths (see Table 7).

TABLE 7 Absorbance (L · g⁻¹ · cm⁻¹) of prior art HSA preparations SAMPLEA₃₅₀ A₄₀₃ A₅₀₀ 1 9.95 × 10⁻³ 4.10 × 10⁻³ 0.8 × 10⁻³ 2 9.25 × 10⁻³ 5.36 ×10⁻³ 1.1 × 10⁻³ 3 7.40 × 10⁻³ 3.26 × 10⁻³ 0.6 × 10⁻³ 4 7.20 × 10⁻³ 3.60× 10⁻³ 0.6 × 10⁻³ 5 8.68 × 10⁻³ 4.08 × 10⁻³ 0.8 × 10⁻³ 6 11.45 × 10⁻³ 6.26 × 10⁻³ 1.2 × 10⁻³ 7 7.20 × 10⁻³ 3.70 × 10⁻³ 0.8 × 10⁻³ 8 6.82 ×10⁻³ 4.78 × 10⁻³ 1.8 × 10⁻³

Endotoxin

A solution of drug product is assayed using Limulus amoebocyte lysate bykinetic turbidimetric measurement at 340 nm, at a temperature of36.5-37.5° C. using a automatic endotoxin detection system (eg, LAL5000E). A standard curve is constructed from known concentrations of astandard endotoxin preparation, negative controls and test materialsolution spiked with a known quantity of standard endotoxin are alsoincluded in the assay. The change in turbidity of the reaction mixtureis measured over time and a log-log regression. Any endotoxin in thedrug product is quantified against the standard curve and recovery ofthe endotoxin spike is confirmed. No endotoxin was detected.

Free Thiol

Ellman's Reagent, 5,5′-Dithiobis-(2-Nitrobenzoate) (DTNB) is a specificmeans of detecting free sulfydryl groups such as cys-SH (Cys-residue 34in the case of rHA). Idle reaction releases the 5 thio-2-nitrobenzoateion TNB²⁻ which has an absorption maximum at 412 nm. By measuring theincrease in absorbance at 412 nm and dividing by the molar extinctioncoefficient of the TNB²⁻ ion at 412 nm, the free sulfydryl content ofrHA can be calculated.

Sample mol · mol⁻¹ A 0.82 B 0.77 C 0.77 D 0.85 E 0.90

1-53. (canceled)
 54. A process for purifying a recombinant albuminsolution, the process comprising— (a) subjecting a first recombinantalbumin solution of pH 8.0-9.5 buffered with a buffer comprising adivalent cation at a concentration of 5-250 mM, and having aconductivity in the range of 1 to 75 mS·cm⁻¹, to an affinitychromatography step which is run in negative mode with respect to thealbumin and which utilises an affinity matrix comprising immobiliseddihydroxyboryl groups, thereby obtaining a purified albumin solution;and (b) subjecting the thus purified albumin solution from step (a) to aconcentration step, a diafiltration step and a cation exchangechromatography step, wherein the diafiltration step is conducted at a pHof 2.5-6.0, wherein the cation exchange step is run in negative modewith respect to albumin and under conditions such that glycosylatedalbumin binds to the cation exchange material, and wherein the albuminsolution which undergoes cation exchange chromatography has an albuminconcentration of 10-250 g·L⁻¹; (c) thereby to produce a finalrecombinant albumin solution having a nickel ion content of less than100 ng per gram of albumin.
 55. The process of claim 54 wherein thediafiltration step is conducted at a pH of 4.0-6.0.
 56. The process ofclaim 54 wherein the diafiltration step is conducted at a pH 5.3±0.5.57. The process of claim 54 wherein the recombinant albumin solution isobtained from a fungal culture medium obtained by culturing a fungustransformed with an albumin-encoding nucleotide sequence in afermentation medium, whereby said fungus expresses albumin and secretesit into the medium.
 58. The process of claim 54 wherein the cationexchange step utilises a matrix which comprises immobilised sulfopropylsubstituents as cation exchangers.
 59. The process of claim 54 whereinthe albumin solution which undergoes cation exchange chromatography hasa pH of 4.5-6.0.
 60. The process of claim 54 wherein the albuminsolution which undergoes cation exchange chromatography has a pH of5.0-5.6.
 61. The process of claim 54 wherein the albumin solution whichundergoes cation exchange chromatography has a pH of 5.2-5.4.
 62. Theprocess of claim 54 wherein the albumin solution which undergoes cationexchange chromatography has an albumin concentration of 20-70 g·L⁻¹. 63.The process of claim 54 wherein the albumin solution which undergoescation exchange chromatography has an albumin concentration of 50±10g·L⁻¹.
 64. The process of claim 54 wherein the albumin solution whichundergoes cation exchange chromatography run in the negative mode withrespect to albumin comprises octanoate and/or another fatty acid. 65.The process of claim 64 wherein the albumin solution which undergoescation exchange chromatography run in the negative mode with respect toalbumin has an octanoate ion concentration of 2-15 mM.
 66. The processof claim 65 wherein the octanoate ion concentration is 5-10 mM.
 67. Theprocess of claim 66 wherein the octanoate ion concentration is 6-9 mM.68. The process of claim 54 wherein prior to the cation exchange stepthe albumin solution undergoes (i) pH-adjustment; and/or (ii)conditioning by addition of octanoate and/or other fatty acid.
 69. Theprocess of claim 54 wherein the pH of the first albumin solution treatedin step (a) of claim 54 is pH 8.0-9.0.
 70. The process of claim 54wherein the pH of the first albumin solution treated in step (a) ofclaim 54 is pH 8.3-pH8.6.
 71. The process of claim 54 wherein the firstalbumin solution treated in step (a) of claim 54 is buffered with abuffer which comprises divalent cation at a concentration of 10-100 mM.72. The process of claim 54 wherein the divalent cation in the buffer ofthe first albumin solution treated in step (a) of claim 54 is calcium.73. The process of claim 72 wherein the calcium is in the form of CaCl₂.74. The process of claim 54 wherein the first albumin solution treatedin step (a) of claim 54 is buffered with a buffer which comprisesglycine at a concentration of 10-500 mM; NaCl at a concentration of0-500 mM; and CaCl₂ at a concentration of 5-250 mM.
 75. The process ofclaim 74 wherein the buffer comprises glycine at a concentration of25-200 mM.
 76. The process of claim 74 wherein the buffer comprisesglycine at a concentration of 50-150 mM.
 77. The process of claim 74wherein the buffer comprises NaCl at a concentration of 25-200 mM. 78.The process of claim 74 wherein the buffer comprises NaCl at aconcentration of 50-150 mM.
 79. The process of claim 74 wherein thebuffer comprises CaCl₂ at a concentration of 10-100 mM.
 80. The processof claim 54 wherein the first albumin solution treated in step (a) ofclaim 54 is buffered with a buffer which comprises about 100 mM glycine,about 100 mM NaCl and about 50 mM CaCl₂.
 81. The process of claim 54wherein the first albumin solution treated in step (a) of claim 54 isbuffered with a buffer having a conductivity of 10-50 mS·cm⁻¹.
 82. Theprocess of claim 54 wherein the first albumin solution treated in step(a) of claim 54 is buffered with a buffer having a conductivity of 18-22mS·cm⁻¹.
 83. The process of claim 54 wherein the first albumin solutiontreated in step (a) of claim 54 comprises albumin at a concentration ofat least 70 g·L⁻¹.
 84. The process of claim 54 wherein the albumin isloaded in step (a) of claim 54 in less than 0.5 column volumes.
 85. Theprocess of claim 54 wherein the albumin is loaded in step (a) of claim54 in less than 0.35 column volumes.
 86. The process of claim 54 whereinthe matrix used in step (a) of claim 54 comprises immobilised boronicacid.
 87. The process of claim 54 wherein the matrix used in step (a) ofclaim 54 comprises aminophenylboronic acid.
 88. The process of claim 54further comprising an anion exchange step, preferably utilising a matrixwhich comprises immobilised dialkylaminoalkyl substituents as anionexchangers, followed by a further diafiltration step that is conductedat a pH of 2.5-6.0.
 89. The process of claim 88 wherein the anionexchange step is run in negative mode with respect to the albumin. 90.The process of claim 88 wherein the anion exchange step is run inpositive mode with respect to the albumin.
 91. The process of claim 89wherein, prior to the anion exchange step, the albumin solutionundergoes one or more of: buffer exchange; concentration; dilution;dialysis; diafiltration; pH-adjustment; treatment with a reducing agent;decolouration treatment; heating; cooling or a non-purifying handlingstep which improves the environment or condition of the albumin for thenext step of the process or for final use.
 92. The process of claim 90wherein, prior to the anion exchange step, the albumin solutionundergoes one or more of: buffer exchange; concentration; dilution;dialysis; diafiltration; pH-adjustment; treatment with a reducing agent;decolouration treatment; heating; cooling or a non-purifying handlingstep which improves the environment or condition of the albumin for thenext step of the process or for final use.
 93. The process of claim 54which is preceded by one or more of the following steps: fermentation;separation of albumin from a recombinant albumin-producing host cell;subjecting a centrate from fermentation to a non-purifying handling stepwhich improves the environment or condition of the albumin for the nextstep of the process or for final use; cation exchange chromatography;anion exchange chromatography; or affinity chromatography.
 94. Theprocess of claim 93, wherein the process of claim 54 is preceded bycation exchange chromatography using sulfopropyl substituents as cationexchangers.
 95. The process of claim 93, wherein the process of claim 54is preceded by anion exchange chromatography using diethylaminoalkylsubstituents as anion exchangers.
 96. The process of claim 93, whereinthe process of claim 54 is preceded by affinity chromatography using anaffinity matrix which comprises an immobilised albumin-specific dye. 97.The process of claim 96 in which the immobilised albumin-specific dye isa Cibacron Blue type of dye.
 98. A process for purifying a recombinantalbumin solution, the process comprising the steps of: (a) subjecting arecombinant albumin solution to a cation exchange chromatography steprun in positive mode with respect to the albumin; (b) collecting analbumin-containing cation exchange eluate; (c) subjecting the cationexchange eluate to an anion exchange chromatography step run in positivemode with respect to the albumin; (d) collecting an albumin-containinganion exchange eluate; (e) subjecting the anion exchange eluate to anaffinity chromatography step run in positive mode with respect to thealbumin; (f) collecting an albumin-containing affinity chromatographyeluate; (g) subjecting the affinity chromatography eluate to an affinitychromatography step run in negative mode with respect to the albumin andin positive mode with respect to glycoconjugates according to step (a)of claim 54; (h) collecting the albumin-containing affinitychromatography flow through; (i) subjecting the affinity chromatographyflow through to concentration, diafiltration and cation exchangechromatography steps according to step (b) of claim 54 to produce arecombinant albumin-containing cation exchange flow through having anickel ion content of less than 100 ng per gram of albumin; (l)collecting the albumin-containing cation exchange flow through; (k)subjecting the cation exchange flow through to an anion exchangechromatography step run in negative mode or positive mode; (l)collecting the albumin-containing anion exchange flow through whereinthe anion exchange step is run in negative mode; or eluting from theanion exchange matrix an anion exchange eluate wherein the anionexchange step is run in positive mode; and (m) a diafiltration step thatis conducted at a pH of 2.5-6.0.
 99. The process of claim 98 wherein anyof steps (a) to (m) are preceded or followed by one or more of: bufferexchange; concentration; dilution; dialysis; diafiltration;pH-adjustment; treatment with a reducing agent; decolouration treatment;heating; cooling or a non-purifying handling step which improves theenvironment or condition of the albumin for the next step of the processor for final use.
 100. A process for purifying a recombinant albuminsolution, the process comprising the steps of: (a) subjecting arecombinant albumin solution to a cation exchange chromatography steprun in positive mode with respect to the albumin; (b) collecting analbumin-containing cation exchange eluate; (c) subjecting the cationexchange eluate to an anion exchange chromatography step run in positivemode with respect to the albumin; (d) collecting an albumin-containinganion exchange eluate; (e) subjecting the anion exchange eluate to anaffinity chromatography step run in positive mode with respect to thealbumin; (f) collecting an albumin-containing affinity chromatographyeluate; (g) subjecting the affinity chromatography eluate to an affinitychromatography step run in negative mode with respect to the albumin andin positive mode with respect to glycoconjugates according to step (a)of claim 54; (h) collecting the albumin-containing affinitychromatography flow through; (i) subjecting the affinity matrix flowthrough to an anion exchange chromatography step run in negative orpositive mode with respect to the albumin; (j) collecting thealbumin-containing anion exchange flow through wherein the anionexchange step is run in negative mode; or eluting from the anionexchange matrix an anion exchange eluate wherein the anion exchange stepis run in positive mode; (k) subjecting the albumin solution purified bythe anion exchange chromatography step to concentration, diafiltrationand cation exchange chromatography steps in accordance with step (b) ofclaim 54 to produce a recombinant albumin-containing cation exchangeflow through having a nickel ion content of less than 100 ng per gram ofalbumin; (l) collecting the albumin-containing cation exchange flowthrough.
 101. The process of claim 100 wherein any of steps (a) to (l)are preceded or followed by one or more of: buffer exchange;concentration; dilution; dialysis; diafiltration; pH-adjustment;addition of reducing agent; decolouration treatment; heating; cooling ora non-purifying handling step which improves the environment orcondition of the albumin for the next step of the process or for finaluse.
 102. The process of claim 54 wherein the recombinant albumin isproduced by a process comprising culturing a fungal cell expressing arecombinant albumin coding sequence and obtaining the albumin, whereinthe cell has a genetic modification in a coding region of a geneencoding a protein O-mannosyltransferase (PMT) enzyme or in a regioninvolved in the expression of a gene encoding a PMT enzyme which causesthe cell to have at least a reduced capacity of mannosylation of therecombinantly-expressed albumin and wherein the culture medium is atleast 1,000 L and is of pH5.3-6.8.
 103. The process of claim 102 whereinsaid modification(s) comprises any suppression, substitution, deletion,addition, disruption and/or mutational insertion.
 104. The process ofclaim 103 wherein said modification(s) are stably-inherited and/or arenon-reverting and/or are non-leaky.
 105. The process of claim 102wherein the gene encoding a PMT enzyme encodes PMT1.
 106. The process ofclaim 102 wherein the fungal cell is cultured in a culture medium of atleast 5,000 L.
 107. The process of claim 106 wherein the fungal cell iscultured in a culture medium of at least 7,500 L.
 108. The process ofclaim 102 wherein the fungal cell is cultured at pH 6.2-6.7.
 109. Theprocess of claim 108 wherein the fungal cell is cultured at pH 6.3-6.5.110. The process of claim 54 wherein the initial albumin solution isderived from a yeast culture medium obtained by culturing yeasttransformed with an albumin-encoding nucleotide sequence in afermentation medium, whereby said yeast expresses albumin and secretesit into the medium.
 111. The process of claim 110 wherein the yeast isof the genus Saccharomyces.
 112. The process of claim 111 wherein theyeast is Saccharomyces cerevisiae.
 113. The process of claim 110 whereinthe yeast is of the genus Pichia or Kluyveromyces.
 114. The process ofclaim 102 wherein the albumin is encoded by a DNA sequence whichcomprises a recombinant albumin coding sequence wherein the 3′ end ofthe recombinant albumin coding sequence comprises two or more in-frametranslation stop codons, and preferably three in-frame translation stopcodons.
 115. The process of claim 110 wherein the albumin is encoded bya DNA sequence which comprises a recombinant albumin coding sequencewherein the 3′ end of the recombinant albumin coding sequence comprisestwo or more in-frame translation stop codons, and preferably threein-frame translation stop codons.
 116. The process of any one of claims54, 88, 98, 99, 100 or 101 wherein the albumin product is formulated forparenteral administration to a human, sterilised or placed into a finalcontainer.